Multiple dosing with viral vectors

Abstract

The present invention relates, at least in part, to repeated lower doses of viral vectors administered in combination with synthetic nanocarriers attached to immunosuppressant drugs, and related compositions.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Application Nos. 63 / 279,174, filed November 14, 2021; 63 / 300,785, filed January 19, 2022; 63 / 317,576, filed March 8, 2022; and 63 / 338,672, filed May 5, 2022; the entire contents of each of which are incorporated herein by reference. Summary of the Invention

[0002] SUMMARY OF THE PRESENT APPLICATION The present invention relates, at least in part, to repeated lower doses of viral vectors and related compositions administered in combination with synthetic nanocarriers attached to immunosuppressants. The compositions and methods provided herein can be used to provide effective transgene expression while sparing doses, for example to provide reduced toxicity and / or manufacturing benefits. In addition, it has been surprisingly found that multiple lower doses of viral vectors in a dosing can be administered in combination with at least one dose of synthetic nanocarriers comprising an immunosuppressant in that dosing (e.g., in combination), at a higher dose (e.g., a single higher dose, e.g., 1e10 14 Vector genomes / kg or 5e 13 It has been found that these vectors can result in transgene expression equivalent to that of viral vectors at 100 ng / kg (dose of vector genomes / kg).

[0003] In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of the viral vector administered is lower than the dose of the viral vector administered to a subject not administered (e.g., co-administration) with a synthetic nanocarrier comprising an immunosuppressant, but achieves the same or equivalent level of transgene expression in a comparable subject or test subject. In one embodiment of any one of the compositions or methods provided herein, the sum of the repeated lower doses of the viral vector administered is less than half the dose of the viral vector administered to a subject not administered (e.g., co-administration) with a synthetic nanocarrier comprising an immunosuppressant, but achieves the same or equivalent level of transgene expression. In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of the viral vector administered is at least 1 / 5 but less than the dose of the viral vector administered to a subject not administered (e.g., co-administration) with a synthetic nanocarrier comprising an immunosuppressant, but achieves the same or equivalent level of transgene expression. In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of the viral vector administered is no more than 1 / 5 of the dose of the viral vector administered to a subject not administered (e.g., co-administered) with a synthetic nanocarrier comprising an immunosuppressant, yet achieves the same or equivalent level of transgene expression. In one embodiment of any one of the compositions or methods provided herein, each of the repeated lower doses of the viral vector administered is no more than 1 / 5 of the dose of the viral vector administered to a subject not administered (e.g., co-administered) with a synthetic nanocarrier comprising an immunosuppressant, yet achieves the same or equivalent level of transgene expression.

[0004] In one embodiment of any one of the compositions or methods provided herein, a lower dose of viral vector administered to a subject is given under the same conditions as a dose of viral vector administered without the synthetic nanocarrier comprising an immunosuppressant drug, but otherwise achieves the same or comparable levels of transgene expression in a comparable subject or test subject. In one embodiment of any one of the compositions or methods provided herein, a lower dose(s) of viral vector is administered monthly or bimonthly in combination with the synthetic nanocarrier comprising an immunosuppressant drug.

[0005] In one embodiment of any one of the compositions or methods provided herein, the lower dose is lower than the dose of the viral vector administered without the concomitant administration of a synthetic nanocarrier attached to an immunosuppressant drug, but results in equivalent transgene expression as the dose of the viral vector administered without the concomitant administration of a synthetic nanocarrier attached to an immunosuppressant drug.

[0006] In one embodiment of any one of the compositions or methods provided herein, a reduction in an undesirable immune (e.g., humoral) response to the viral vector, and / or effective expression of the transgene or nucleic acid material, and / or durable expression of the transgene or nucleic acid material, and / or equivalent transgene expression, is a result of the dosing(s).

[0007] In one aspect, a method of manufacturing any one of the compositions or kits provided herein is provided. In one embodiment, the method of manufacturing includes producing one or more doses or dosage forms of a viral vector and producing one or more doses or dosage forms of a population of synthetic nanocarriers attached to an immunosuppressant drug. In another embodiment of any one of the methods of manufacturing provided, the step of producing one or more doses or dosage forms of a population of synthetic nanocarriers attached to an immunosuppressant drug includes attaching the immunosuppressant drug to the synthetic nanocarriers. In another embodiment of any one of the methods of manufacturing provided, the method further includes combining one or more doses or dosage forms of a population of synthetic nanocarriers attached to an immunosuppressant drug with one or more doses or dosage forms of a viral vector in the kit.

[0008] In another aspect, there is provided a use of any one of the compositions or kits provided herein for the manufacture of a medicament for reducing or inhibiting an undesired immune response to a viral vector in a subject and / or for providing a persistent and / or effective expression of a transgene or nucleic acid material. In one embodiment, the composition or kit comprises one or more doses or dosage forms comprising a population of synthetic nanocarriers attached to an immunosuppressant drug, and / or one or more doses or dosage forms comprising a viral vector, wherein the population of synthetic nanocarriers attached to an immunosuppressant drug and / or the viral vector are administered according to any one of the methods provided herein. In some embodiments of any one of the uses provided herein, the population of synthetic nanocarriers attached to an immunosuppressant drug does not comprise a viral vector antigen-presenting cell (APC) presentable antigen of the viral vector.

[0009] In another aspect, any one of the compositions or kits provided herein is provided for use in any one of the methods provided herein. [Brief description of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows the expression of viral vectors and ImmTOR (PLA / PLA-PEG synthetic nanocarriers containing rapamycin) at different doses. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Detailed Description of the Invention Before describing the invention in detail, it is to be understood that the invention is not limited to specifically exemplified materials or process parameters, which may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments of the invention only, and is not intended to limit the use of alternative terminology to describe the invention.

[0012] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety for all purposes.

[0013] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to a "polymer" includes a mixture of two or more such molecules or a mixture of different molecular weights of a single polymer type, reference to a "synthetic nanocarrier" includes a mixture of two or more such synthetic nanocarriers or a plurality of such synthetic nanocarriers, reference to a "DNA molecule" includes a mixture of two or more such DNA molecules or a plurality of such DNA molecules, reference to an "immunosuppressant drug" includes a mixture of two or more such materials or a plurality of immunosuppressant drug molecules, etc.

[0014] As used herein, the term "comprise" or variations thereof, such as "comprises" or "comprising," should be read to indicate the inclusion of any recited integer (e.g., a feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., a feature, element, characteristic, property, method / process step, or limitation) but not the exclusion of any other integer or group of integers. Thus, as used herein, the term "comprising" is inclusive and does not exclude additional, unrecited integers or method / process steps.

[0015] In any one of the embodiments of the compositions and methods provided herein, "comprising" may be replaced with "consisting essentially of" or "consisting of." The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps, as well as those that do not substantially affect the characteristics or function of the claimed invention. As used herein, the term "consisting" is used to indicate that the recited integer (e.g., a feature, element, characteristic, property, method / process step, or limitation) or group of integers (e.g., a feature, element, characteristic, property, method / process step, or limitation) is present alone.

[0016] A. Introduction Certain dosing provided herein may result in equivalent, effective, and / or permanent expression of the transgene or nucleic acid material. Dosing may also, in some embodiments, result in a dose-sparing effect.

[0017] B. Definition "Administering" or "administration" or "administering" means providing a material to a subject in a pharmacologically useful manner. The term is intended in some embodiments to encompass "causing to be administered." "Causing to be administered" includes directly or indirectly causing, inciting, encouraging, assisting, inducing, or directing another party to administer the material.

[0018] "Administration schedule" refers to the administration of one or more drug dosages according to a determined schedule. The schedule may include the number of dosages as well as the frequency of such dosages or the interval between dosages. Such administration schedules may include a number of parameters that are varied to achieve a specific goal, preferably a reduction in undesired immune responses to viral vector antigens, and / or effective and / or persistent and / or equivalent expression of the transgene or nucleic acid material. In embodiments, the administration schedule is any of the administration schedules as provided below in the examples. In some embodiments, an administration schedule according to the present invention may be used to administer the dosage to one or more test subjects. The immune responses and / or expression of the transgene or nucleic acid material in these test subjects may then be assessed to determine whether the schedule was effective in reducing undesired immune responses and / or effective and / or persistent and / or equivalent expression of the transgene or nucleic acid material. Whether the schedule had the desired effect may be determined using any of the methods provided herein or otherwise known in the art. For example, samples may be obtained from subjects to whom the dosages provided herein have been administered according to a particular dosing schedule to determine whether a particular immune cell cytokine, antibody, etc., has been reduced, produced, activated, etc., and / or whether a particular protein or expression product has been increased, reduced, produced, etc. Methods useful for detecting the presence and / or number of immune cells include, but are not limited to, flow cytometry methods (e.g., FACS), ELISpot, proliferative responses, cytokine production, and immunohistochemical methods. Methods useful for determining the production levels of proteins such as antibodies are well known in the art and include the assays provided herein. Such assays include ELISA assays.

[0019] The transgene expression level can be assessed in a comparable subject or a test subject. Those skilled in the art will understand that a "comparable subject" is a subject whose expression level can be determined and used as a comparator with the subject to be treated. A "test subject" is any subject whose expression level can be determined and whose level can be a direct or indirect comparison standard, for example, through scaling and / or extrapolation. In some embodiments, the lower dose is determined by comparing the candidate lower dose in the comparable subject or test subject with the higher dose administered to the comparable subject or test subject. The dose for the subject to be treated can then be determined through scaling or extrapolation.

[0020] In some embodiments, the lower dose is lower than the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug, but results in expression of the transgene or nucleic acid material equivalent to the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug. The lower dose can be a single lower dose with the concomitant administration of the synthetic nanocarrier with the immunosuppressant drug, or more than one such lower dose over a finite period (e.g., one week or two weeks) with the concomitant administration of at least one synthetic nanocarrier attached to the immunosuppressant drug that results in expression of the equivalent transgene or nucleic acid material. As used herein, "equivalent expression of the transgene or nucleic acid material" refers to expression that has been determined to be not statistically significantly different or would not be expected to result in a significant clinically different effect.

[0021] "Effective amount" in the context of a composition or dosage form for administration to a subject refers to the amount of the composition or dosage form that produces one or more desired immune responses or effective and / or persistent and / or equivalent expression of transgene or nucleic acid material in the subject.Thus, in some embodiments, an effective amount is the amount of any of the compositions or dosage forms provided herein, or combinations thereof, that reduces undesired immune responses and / or provides effective and / or persistent and / or equivalent expression of transgene or nucleic acid material.This amount can be for in vitro or in vivo purposes.For in vivo purposes, the amount can be the amount that a clinician would consider to have clinical benefit for the subject as provided herein.

[0022] An effective amount may involve reducing an undesired immune response level, but in some embodiments, it ultimately involves preventing an undesired immune response. An effective amount may also involve delaying the onset of an undesired immune response. An effective amount may also be an amount that produces a desired therapeutic endpoint or a desired therapeutic effect. An effective amount preferably results in a reduction of an undesired immune response specific to the viral vector in the subject, and / or provides an effective and / or persistent and / or equivalent expression of the transgene or nucleic acid material of the viral vector. An effective amount may also result in a tolerogenic immune response in the subject to an antigen, such as a viral vector antigen. In other embodiments, an effective amount may involve enhancing a desired response level, such as a therapeutic endpoint or outcome. Achievement of any of the above may be monitored by routine methods.

[0023] In some embodiments of any one of the compositions and methods provided, an effective amount is an amount that causes a desired immune response to persist in a subject for at least one week, at least two weeks, at least one month, or longer. In other embodiments of any of the compositions and methods provided, an effective amount is an amount that causes a measurable desired response to be produced, such as a reduced humoral immune response (e.g., the response to a specific antigen), and / or an expression response of a transgene or nucleic acid material, for at least one week, at least two weeks, at least one month, or longer.

[0024] The effective amount will, of course, depend on the particular subject being treated; the severity of the condition, disease, or disorder; individual patient parameters, including age, physical condition, size, and weight; duration of treatment; the nature of concurrent treatment (if any); the particular route of administration, and similar factors within the knowledge and experience of the health practitioner. These factors are well known to those of skill in the art and can be addressed with a little routine experimentation. It is generally preferred to use a maximum dose, that is, the highest safe dose following sound medical judgment. However, it will be understood by those skilled in the art that a patient may require a lower dose or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0025] The dose of a synthetic nanocarrier attached to an immunosuppressant drug may refer to the amount of immunosuppressant drug attached to the synthetic nanocarrier. Alternatively, the dose may be administered based on the number of synthetic nanocarriers that provide the desired amount of immunosuppressant drug.

[0026] "Anti-viral vector immune response" or "immune response to a viral vector" or the like refers to any undesired immune response to a viral vector. In some embodiments, the undesired immune response is an antigen-specific immune response to the viral vector or an antigen thereof. In some embodiments, the immune response is specific to a viral antigen of the viral vector. The immune response may be an anti-viral vector antibody response, an anti-viral vector T cell immune response, such as a CD4+ T cell or CD8+ T cell immune response, or an anti-viral vector B cell immune response.

[0027] "Antigen" refers to a B cell antigen or a T cell antigen. "Type(s) of antigen" refers to molecules that share the same or substantially the same antigenic characteristics. In some embodiments, the antigen may be a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, polysaccharide, or may be contained or expressed in a cell. In some embodiments, such as when the antigen is not well defined or characterized, the antigen may be contained within a cell or tissue preparation, cell debris, cell exosomes, conditioned medium, etc.

[0028] "Antigen-specific" refers to any immune response that occurs as a result of the presence of an antigen or a portion thereof, or that generates a molecule that specifically recognizes or binds to the antigen. In some embodiments, when the antigen is of a viral vector, antigen-specific may mean viral vector-specific. For example, when the immune response is antigen-specific antibody production, such as viral vector-specific antibody production, an antibody that specifically binds to the antigen (e.g., the viral vector) is produced. As another example, when the immune response is antigen-specific B cell or CD4+ T cell proliferation and / or activity, the proliferation and / or activity occurs as a result of recognition of the antigen or a portion thereof, alone or in complex with MHC molecules, B cells, etc.

[0029] "Assessing an immune response" refers to any measurement or determination of the level, presence or absence, reduction, increase, etc., of an immune response in vitro or in vivo. Such measurements or determinations may be performed on one or more samples obtained from a subject. Such assessments may be performed by any of the methods provided herein or otherwise known in the art.

[0030] "Attach" or "Attached" or "Couple" or "Coupled" (and the like) means to chemically associate one entity (e.g., moiety) with another. In some embodiments, the attachment is covalent, meaning that the attachment occurs in the context of the presence of a covalent bond between the two entities. In non-covalent embodiments, the non-covalent attachment is mediated by non-covalent interactions, including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-object interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. In embodiments, encapsulation is a form of attachment. In embodiments, the viral vector and the synthetic nanocarrier attached to the immunosuppressant drug are not attached to one another, meaning that the viral vector and the synthetic nanocarrier attached to the immunosuppressant drug have not been subjected to a process specifically intended to chemically link one to the other.

[0031] A "at risk" subject is one who a health care practitioner believes may have a disease, disorder, or illness, or who a health care practitioner believes may experience an undesirable immune response as provided herein and would benefit from the compositions and methods provided. In some embodiments, the subject is one who would benefit from administration of a viral vector. "Average," as used herein, unless otherwise specified, refers to the arithmetic mean.

[0032] "Concomitantly" means administering two or more materials / agents to a subject in a time-correlated manner, preferably sufficiently time-correlated to provide modulation of immune or physiological responses, and even more preferably, two or more materials / agents are administered in combination. In embodiments, concomitant administration may cover administration of two or more materials / agents within a specified period, preferably within a month, more preferably within a week, even more preferably within a day, and even more preferably within an hour. In embodiments, materials / agents may be administered repeatedly in combination, which is concomitant administration on more than one occasion, for example as may be provided in the examples.

[0033] "Determining" or "determining" means to ascertain a factual situation. Determining may be accomplished in a number of ways, including, but not limited to, conducting an experiment or making a prediction. By way of illustration, the dose of the immunosuppressant drug and / or viral vector may be determined by starting with a test dose and using known scaling techniques (such as allometric or isometric scaling) to determine the dose for administration. Such may also be used to determine the protocol or administration schedule as provided herein. In another embodiment, the dose may be determined by testing various doses in a subject, i.e., through direct experimentation based on experience and guiding data. In an embodiment, "determining" or "determining" includes "causing to be determined". "Determining" means to cause, encourage, encourage, assist, induce, or direct or act in concert with an entity to ascertain a factual situation; including directly or indirectly, or explicitly or implicitly.

[0034] "Dose" refers to a specific amount of pharmacologically and / or immunologically active material to be administered to a subject for a given period of time. Generally, the dose of synthetic nanocarriers and / or viral vectors comprising an immunosuppressant in the methods and compositions of the present invention refers to the amount of synthetic nanocarriers and / or viral vectors comprising an immunosuppressant. Alternatively, the dose may be administered based on the number of synthetic nanocarriers that provide the desired amount of the immunosuppressant, in the instance where a dose of synthetic nanocarriers comprising an immunosuppressant is referred to. When dose is used in the context of repeated dosing, the dose refers to the amount of each of the repeated doses, which may be the same or different.

[0035] "Administering" refers to the administration of a pharmacologically and / or immunologically active material or a combination of pharmacologically and / or immunologically active materials to a subject. The materials in the administration may be administered in combination in any one of the methods provided herein. The materials in the administration may be administered separately in separate compositions in any one of the methods provided herein.

[0036] "Encapsulate" means to surround at least a portion of a substance within a synthetic nanocarrier. In some embodiments, the substance is completely enclosed within the synthetic nanocarrier. In other embodiments, most or all of the encapsulated substance is not exposed to the local environment outside the synthetic nanocarrier. In other embodiments, no more than 50%, 40%, 30%, 20%, 10%, or 5% (weight / weight) is exposed to the local environment. Encapsulation is distinct from absorption, which places most or all of the substance on the surface of the synthetic nanocarrier, leaving the substance exposed to the local environment outside the synthetic nanocarrier. In any one of the methods or compositions provided herein, the immunosuppressant drug may be encapsulated in the synthetic nanocarrier.

[0037] An "expression control sequence" is any sequence that can affect expression and can include promoters, enhancers, and operators. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a promoter. In one embodiment of any one of the methods or compositions provided, the expression control sequence is a liver-specific promoter or a constitutive promoter. A "liver-specific promoter" is one that provides expression exclusively or preferentially in cells of the liver. A "constitutive promoter" is one that is generally active and does not appear to be exclusive or preferential in a cell. In any one of the nucleic acids or viral vectors provided herein, the promoter can be any one of the promoters provided herein.

[0038] "Generating" means causing an effect, such as an immune response or a physiological response (eg, a tolerogenic immune response), that occurs either directly or indirectly itself.

[0039] "Identifying a subject" is any action or course of action that allows a clinician to recognize a subject as a subject that will benefit from the method, composition, or kit provided herein. Preferably, the identified subject is a subject that needs therapeutic benefit from a viral vector and is expected to experience an undesirable immune response as provided herein. The action or course of action may itself be either direct or indirect. In one embodiment of any one of the methods provided herein, the method further comprises identifying a subject that needs the method, composition, or kit provided herein.

[0040] "Immunosuppressant" refers to a compound that causes APC to have an immunosuppressive effect (e.g., a tolerogenic effect) or suppress T cells or B cells. An immunosuppressive effect generally refers to the production or expression of cytokines or other factors by APC that reduce, inhibit, or prevent undesired immune responses, or promote desired immune responses, such as regulatory immune responses. When an APC acquires (under an immunosuppressive effect) an immunosuppressive function on immune cells that recognize the antigen presented by the APC, the immunosuppressive effect is said to be specific to the antigen presented. Without being bound by any particular theory, it is believed that the immunosuppressive effect is the result of the immunosuppressant being delivered to the APC, preferably in the presence of the antigen. In one embodiment, the immunosuppressant causes APC to promote a regulatory phenotype in one or more immune effector cells. For example, the regulatory phenotype may be characterized by the inhibition of the production, induction, stimulation, or mobilization of antigen-specific CD4+ T cells or B cells, the inhibition of the production of antigen-specific antibodies, the production, induction, stimulation, or mobilization of Treg cells (e.g., CD4+CD25highFoxP3+ Treg cells), and the like. This may be the result of the conversion of CD4+ T cells or B cells to a regulatory phenotype. This may also be the result of FoxP3 induction in other immune cells, such as CD8+ T cells, macrophages, and iNKT cells. In one embodiment, the immunosuppressant is one that affects the response after APCs process antigen. In another embodiment, the immunosuppressant is not one that interferes with antigen processing. In a further embodiment, the immunosuppressant is not an apoptotic signaling molecule. In another embodiment, the immunosuppressant is not a phospholipid.

[0041] Immunosuppressants include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase inhibitors, such as trichostatin A; corticosteroids; inhibitors of mitochondrial function, such as rotenone; P38 inhibitors; NF-κβ inhibitors, such as 6Bio, dexamethasone, TCPA-1, and IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE2), such as misoprostol; phosphodiesterase inhibitors, such as phosphodiesterase 4 inhibitors (PDE4), such as rolipram; proteasome inhibitors; kinase inhibitors; G protein-coupled receptor agonists; G protein-coupled receptor antagonists; glucocorticoids; retinoids; cytokine inhibitors; cytokine receptor inhibitors; These include oxidized ATP such as cytokine receptor activators; peroxisome proliferator-activated receptor antagonists; peroxisome proliferator-activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; phosphatase inhibitors; PI3KB inhibitors such as TGx-221; autophagy inhibitors such as 3-methyladenine; aryl hydrocarbon receptor inhibitors; proteasome inhibitor I (PSI); and P2x receptor blockers. Immunosuppressants also include IDO, vitamin D3, cyclosporines such as cyclosporine A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine (AZA), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin A, salmeterol, mycophenolate mofetil (MMF), aspirin and other COx inhibitors, niflumic acid, estriol, methotrexate, and triptolide. In embodiments, immunosuppressants may include any of the agents provided herein.

[0042] An immunosuppressant can be a compound that provides an immunosuppressive effect directly on APCs, or it can be a compound that provides an immunosuppressive effect indirectly (i.e., after being processed in some way after administration). Immunosuppressants therefore include prodrug forms of any of the compounds provided herein.

[0043] In one embodiment of any of the methods, compositions, or kits provided herein, the immunosuppressant drug provided herein is attached to the synthetic nanocarrier. In a preferred embodiment, the immunosuppressant drug is an additional element to the material that makes up the structure of the synthetic nanocarrier. For example, in one embodiment, when the synthetic nanocarrier is made up of one or more polymers, the immunosuppressant drug is a compound that is attached to the one or more polymers in addition to the one or more. As another example, in one embodiment, when the synthetic nanocarrier is made up of one or more lipids, the immunosuppressant drug is also a compound that is attached to the one or more lipids in addition to the one or more. In some embodiments, such as when the material of the synthetic nanocarrier also provides an immunosuppressive effect, the immunosuppressant drug is an additional element present in the material of the synthetic nanocarrier that provides the immunosuppressive effect.

[0044] Other exemplary immunosuppressants include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies against CD20, CD3, CD4), biologic-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAI, antisense nucleic acids, aptamers, methotrexate, NSAIDs; fingolimod; natalizumab; alemtuzumab; anti-CD3; tacrolimus (FK506); cytokines and growth factors such as TGF-β and IL-10; and the like. Additional immunosuppressants will be known to those of skill in the art, and the invention is not limited in this respect.

[0045] "Load" is the amount (wt / wt) of immunosuppressant drug attached to a synthetic nanocarrier based on the total dry formulation weight of the material in the total synthetic nanocarrier when attached to the synthetic nanocarrier. Generally, such loading is calculated as an average across a population of synthetic nanocarriers. In one embodiment, the immunosuppressant drug loading is between 0.1% and 99%, on average across the synthetic nanocarriers. In another embodiment, the loading is between 0.1% and 50%. In yet another embodiment, the immunosuppressant drug loading is between 0.1% and 20%. In a further embodiment, the immunosuppressant drug loading is between 0.1% and 10%. In yet a further embodiment, the immunosuppressant drug loading is between 1% and 10%. In yet a further embodiment, the immunosuppressant drug loading is between 7% and 20%. In yet another embodiment, the immunosuppressant drug loading is at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, or at least 20%, at least 25%, or at least 30%, on average across the entire population of synthetic nanocarriers. In yet further embodiments, the immunosuppressant loading is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% on average across the population of synthetic nanocarriers. In some of the above embodiments, the immunosuppressant loading is 25% or less or 30% or less on average across the population of synthetic nanocarriers. In some embodiments, the loading is calculated as may be described in the Examples or as otherwise known in the art.

[0046] "Maximum dimension of a synthetic nanocarrier" means the maximum dimension of a nanocarrier measured along any axis of the synthetic nanocarrier. "Minimum dimension of a synthetic nanocarrier" means the minimum dimension of a synthetic nanocarrier measured along any axis of the synthetic nanocarrier. For example, for a spherical synthetic nanocarrier, the maximum and minimum dimensions of the synthetic nanocarrier will be substantially the same and will be the size of its diameter. Similarly, for a cuboidal synthetic nanocarrier, the minimum dimension of the synthetic nanocarrier will be the smallest of its height, width, or length, while the maximum dimension of the synthetic nanocarrier will be the largest of its height, width, or length. In one embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm. In one embodiment, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 5 μM. Preferably, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is greater than 110 nm, more preferably greater than 120 nm, more preferably greater than 130 nm, and more preferably still greater than 150 nm. The maximum dimension and aspect ratio of the minimum dimension of the synthetic nanocarriers may vary depending on the embodiment. Illustratively, the aspect ratio of the maximum dimension to the minimum dimension of the synthetic nanocarriers may vary from 1:1 to 1,000,000:1, preferably from 1:1 to 100,000:1, more preferably from 1:1 to 10,000:1, more preferably from 1:1 to 1000:1, even more preferably from 1:1 to 100:1, and even more preferably from 1:1 to 10:1.Preferably, the maximum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or less than 3 μm, more preferably equal to or less than 2 μm, more preferably equal to or less than 1 μm, more preferably equal to or less than 800 nm, more preferably equal to or less than 600 nm, and more preferably still equal to or less than 500 nm. In a preferred embodiment, the minimum dimension of at least 75%, preferably at least 80%, more preferably at least 90% of the synthetic nanocarriers in a sample, based on the total number of synthetic nanocarriers in the sample, is equal to or greater than 100 nm, more preferably equal to or greater than 120 nm, more preferably equal to or greater than 130 nm, more preferably equal to or greater than 140 nm, and more preferably still equal to or greater than 150 nm. Measurements of the dimensions (e.g., effective diameter) of synthetic nanocarriers may be obtained in some embodiments by suspending the synthetic nanocarriers in a liquid (mostly aqueous) medium and using dynamic light scattering (DLS) (e.g., using a Brookhaven ZetaPALS instrument). For example, a suspension of synthetic nanocarriers may be diluted from an aqueous buffer into purified water to achieve a final synthetic nanocarrier suspension concentration of approximately 0.01-0.1 mg / mL. The diluted suspension may be prepared directly in or transferred to a cuvette suitable for DLS analysis. The cuvette may then be placed in the DLS, equilibrated to a controlled temperature, and then scanned for a time sufficient to obtain a stable and reproducible distribution based on appropriate inputs for the viscosity of the medium and the refractive index of the sample. The effective diameter or the average value of the distribution is then reported. Determining the effective size of high aspect ratio or non-spherical synthetic nanocarriers may require augmentative techniques, such as electron microscopy, to obtain a more accurate measurement."Dimension" or "size" or "diameter" of a synthetic nanocarrier refers to the mean value of the particle size distribution, e.g., as obtained using dynamic light scattering.

[0047] "Non-methoxy terminated polymer" refers to a polymer having at least one terminus terminating in a moiety other than methoxy. In some embodiments, the polymer has at least two termini terminating in a moiety other than methoxy. In other embodiments, the polymer does not have any termini terminating in methoxy. "Non-methoxy terminated Pluronic polymer" refers to a polymer other than a linear Pluronic polymer with methoxy at both ends. Polymeric nanoparticles as provided herein may comprise a non-methoxy terminated polymer or a non-methoxy terminated Pluronic polymer.

[0048] "Pharmaceutically acceptable excipient" or "pharmacologically acceptable carrier" refers to a pharmacologically inactive material used together with a pharmacologically active material to formulate a composition. Pharmaceutically acceptable excipients include various materials known in the art, including, but not limited to, sugars (e.g., glucose, lactose, etc.), preservatives such as antimicrobial agents, reconstitution aids, colorants, saline (such as phosphate buffered saline), and buffers.

[0049] "Providing" means an action or series of actions performed by an individual that supplies an item or series of items or a method necessary for the practice of the invention. The action or series of actions may be taken by itself either directly or indirectly.

[0050] "Providing a subject" is any action or course of actions that allows a clinician to contact a subject and administer a composition provided herein to it or perform a method provided herein on it. In some embodiments, the subject is a subject in need of viral vector administration and antigen-specific immune tolerance thereto, or any one of the desired results as provided herein. The action or course of actions may be taken by itself either directly or indirectly. In one embodiment of any one of the methods provided herein, the method further comprises providing a subject.

[0051] "Subject" refers to animals, including warm-blooded mammals such as humans and primates; birds; domestic household or farm animals such as cats, dogs, sheep, goats, cows, horses, and pigs; laboratory animals such as mice, rats, and guinea pigs; fish; reptiles; zoo and wild animals; etc. As used herein, a subject may be a subject in need of any one of the methods or compositions provided herein.

[0052] "Synthetic nanocarrier(s)" refers to a discrete object not found in nature and possessing at least one dimension less than or equal to 5 microns in size. Albumin nanoparticles are generally included as synthetic nanocarriers, however, in some embodiments, synthetic nanocarriers do not include albumin nanoparticles. In embodiments, synthetic nanocarriers do not include chitosan. In other embodiments, synthetic nanocarriers are not lipid-based nanoparticles. In further embodiments, synthetic nanocarriers do not include phospholipids.

[0053] Synthetic nanocarriers may be, but are not limited to, one or more lipid-based nanoparticles (also referred to herein as liquid nanoparticles, i.e., nanoparticles whose structural material is predominantly lipid), polymeric nanoparticles, metal nanoparticles, surfactant-based emulsions, dendrimers, buckyballs, nanowires, virus-like particles (i.e., particles that are primarily composed of viral structural proteins but are not infectious or have low infectivity), peptide or protein-based particles (also referred to herein as protein particles, i.e., particles whose structural material is predominantly peptides or proteins) (such as albumin nanoparticles), and / or nanoparticles developed using a combination of nanomaterials, such as lipid-polymer nanoparticles. Synthetic nanocarriers may be of a variety of different shapes, including, but are not limited to, spherical, cubic, conical, rectangular, cylindrical, doughnut-shaped, and the like. Synthetic nanocarriers according to the present invention comprise one or more surfaces. Exemplary synthetic nanocarriers that may be adapted for use in the practice of the present invention include: (1) Gref et al. ofNo. 5,543,158, (2) the polymeric nanoparticles of Published U.S. Patent Application No. 20060002852 to Saltzman et al., (3) the lithographically constructed nanoparticles of Published U.S. Patent Application No. 20090028910 to DeSimone et al., (4) the nanoparticles disclosed in International Publication No. WO 2009 / 051837 to von Andrian et al., (5) the nanoparticles disclosed in Published U.S. Patent Application No. 2008 / 0145441 to Penades et al., (6) the nanoparticles disclosed in U.S. Patent Application No. 2008 / 0145441 to de los (7) the virus-like particles disclosed in published U.S. patent application No. 200602226525 by Rios et al., (8) the nucleic acid-attached virus-like particles disclosed in published U.S. patent application No. 20060251677 by Bachmann et al., (9) the virus-like particles disclosed in WO2010047839A1 or WO2009106999A2, (10) P. Paolicelli et al., “Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Nanoparticles” (11) the apoptotic cells, apoptotic bodies, or synthetic or semi-synthetic mimetics disclosed in U.S. Publication No. 2002 / 0086049; or (12) those of Look et al., Nanogel-based delivery of mycophenolic acid ameliorates systemic lupus erythematosus in mice” J. Clinical Investigation 123(4):1741-1749 (2013). In embodiments, the synthetic nanocarriers may possess an aspect ratio greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0054] Synthetic nanocarriers according to the invention having a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not include a surface with hydroxyl groups that activate complement, or alternatively, include a surface that essentially consists of moieties that are not hydroxyl groups that activate complement. In a preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not include a surface that substantially activates complement, or alternatively, include a surface that essentially consists of moieties that do not substantially activate complement. In a more preferred embodiment, synthetic nanocarriers according to the invention having a minimum dimension equal to or less than about 100 nm, preferably equal to or less than 100 nm, do not include a surface that substantially activates complement, or alternatively, include a surface that essentially consists of moieties that do not activate complement. In an embodiment, the synthetic nanocarriers exclude virus-like particles. In embodiments, synthetic nanocarriers may possess an aspect ratio greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or greater than 1:10.

[0055] "Expression of a transgene or nucleic acid material" refers to the level of expression product of a transgene or nucleic acid material of a viral vector in a subject when the transgene or nucleic acid material is delivered by the viral vector. In some embodiments, the level of expression may be determined by measuring the transgene protein concentration in various tissues or systems of interest in a subject, a matched subject, or a test subject, or between two sets of matched subjects or between two sets of test subjects. Alternatively, when the expression product is a nucleic acid, the level of expression may be measured by the nucleic acid product. Increased expression may be determined, for example, by measuring the amount of expression product in a sample obtained from a subject and comparing it to a previous sample. Persistence of expression may be measured by similar or other methods that will be apparent to one of skill in the art. The sample may be a tissue sample. In some embodiments, the expression product may be measured using flow cytometry.

[0056] "Unwanted immune response" refers to any undesired immune response that results from exposure to an antigen, promotes or exacerbates a disease, disorder, or condition (or a symptom thereof) provided herein, or is indicative of a symptom of a disease, disorder, or condition provided herein. Such immune responses may generally have a negative impact on the subject's health status or may be indicative of a symptom of a negative impact on the subject's health status. Undesired immune responses may be undesired humoral immune responses that may include antigen-specific antibody production, antigen-specific B cell proliferation and / or activity, or antigen-specific CD4+ T cell proliferation and / or activity. Generally, as used herein, these undesired immune responses are specific to the viral vector and antagonize the desired beneficial effects of administration with the viral vector.

[0057] "Viral vector" refers to a vector construct with viral components such as capsid and / or coat proteins that are adapted to contain and deliver a transgene or nucleic acid material (which transgene or nucleic acid material can be expressed as provided herein) encoding a therapeutic agent such as a therapeutic protein or nucleic acid. "Expressed" or "expression" or the like refers to the synthesis of a functional (i.e., physiologically active for a desired purpose) product after the transgene or nucleic acid material is transduced into a cell and processed by the transduced cell. Such products are also referred to herein as "expression products." Viral vectors can be based on adeno-associated viruses, such as, without limitation, AAV8 or AAV2. Thus, the AAV vectors provided herein are viral vectors based on AAV, such as AAV8 or AAV2, that have viral components such as capsid and / or coat proteins that can be packaged to deliver a transgene or nucleic acid material. In some embodiments, the viral vector is a "chimeric viral vector." In such embodiments, this means that the viral vector is made up of viral components derived from more than one virus or viral vector.

[0058] "Viral vector antigen" refers to an antigen associated with a viral vector (i.e., a viral vector or a fragment thereof that can generate an immune response against the viral vector (e.g., production of anti-viral vector-specific antibodies). A viral vector antigen may be presented for recognition by the immune system (e.g., cells of the immune system, including but not limited to, dendritic cells, B cells, or macrophages, such as presented by antigen-presenting cells). A viral vector antigen may be presented, for example, for recognition by T cells. Such antigens may be recognized by and trigger an immune response in T cells through presentation of epitopes of the antigen bound to class I or class II major histocompatibility complex molecules (MHC). Viral vector antigens generally include proteins, polypeptides, peptides, polynucleotides, etc., or are contained or expressed in, on, or by cells. A viral vector antigen, in some embodiments, includes MHC class I-restricted epitopes and / or MHC class II-restricted epitopes and / or B-cell epitopes. In some embodiments, use of the methods, compositions, or kits provided herein results in one or more tolerogenic immune responses specific to the viral vector, hi embodiments, the population of synthetic nanocarriers does not include any added viral vector antigens, meaning that no substantial amount of viral vector antigens has been intentionally added to the synthetic nanocarriers during their manufacture.

[0059] C. Compositions Useful in the Practice of the Methods The transgene or nucleic acid material, e.g., the transgene or nucleic acid material of a viral vector provided herein, may code for any protein or part thereof, or any nucleic acid or part thereof, that is beneficial to a subject, e.g., a subject with a disease or disorder. In an embodiment, the subject has or is suspected of having a disease or disorder due to the endogenous version of the subject's protein being defective or produced in limited amounts or not at all. The subject may have any one of the diseases or disorders as provided herein, and the transgene or nucleic acid material encodes any one of the therapeutic proteins or parts thereof as provided herein. The transgene or nucleic acid material provided herein may code for a functional version of any protein that results in a disease or disorder in the subject through some defect in its endogenous version in the subject, including a defect in the expression of the endogenous version.

[0060] The sequence of the transgene or nucleic acid material may also include expression control sequences. Expression control sequences include promoters, enhancers, and operators, and are generally selected based on the expression system in which the expression construct is to be utilized. In some embodiments, promoter and enhancer sequences are selected for their ability to increase gene expression, while operator sequences may be selected for their ability to regulate gene expression. The transgene may also include sequences that facilitate, and preferably promote, homologous recombination in the host cell. The transgene may also include sequences necessary for replication in the host cell.

[0061] Exemplary expression control sequences include liver-specific promoter sequences and constitutive promoter sequences, such as any one of the promoters provided herein. Generally, the promoter is operatively linked upstream (i.e., 5') of the sequence that codes for the desired expression product. The transgene may also include a suitable polyadenylation sequence operatively linked downstream (i.e., 3') of the coding sequence.

[0062] Viruses have evolved specialized mechanisms for transporting their genomes inside the cells they infect; viral vectors based on such viruses can be tailored to transduce cells. Examples of viral vectors that can be used as provided herein are known in the art or described herein. Suitable viral vectors include, for example, adeno-associated virus (AAV)-based vectors.

[0063] The viral vector provided herein can be based on adeno-associated virus (AAV).AAV vectors are of particular interest for use in therapeutic applications, such as those described herein.AAV is a DNA virus that is not known to cause human disease.Generally, AAV requires co-infection with helper virus (e.g., adenovirus or herpes virus) or expression of helper genes for efficient replication. For a description of AAV-based vectors, see, e.g., U.S. Patent Nos. 8,679,837, 8,637,255, 8,409,842, 7,803,622, and 7,790,449, as well as U.S. Publication Nos. 20150065562, 20140155469, 20140037585, 20130096182, 20120100606, and 20070036757. The AAV vector may be a recombinant AAV vector. The AAV vector can also be a self-complementary (sc) AAV vector, which is described, for example, in U.S. Patent Application Publication Nos. 2007 / 01110724 and 2004 / 0029106, and U.S. Patent Nos. 7,465,583 and 7,186,699.

[0064] The adeno-associated virus on which the viral vector is based may be of a particular serotype, such as AAV8 or AAV2. In some embodiments of any one of the methods or compositions provided herein, the AAV vector is thus an AAV8 vector or an AAV2 vector.

[0065] A wide variety of synthetic nanocarriers can be used to attach the immunosuppressant to the dosage. In some embodiments, the synthetic nanocarriers are spheres or spherical. In some embodiments, the synthetic nanocarriers are flat or planar. In some embodiments, the synthetic nanocarriers are cubes or cubic. In some embodiments, the synthetic nanocarriers are ovals or ellipses. In some embodiments, the synthetic nanocarriers are cylinders, cones, or pyramids.

[0066] In some embodiments, it is desirable to use a population of synthetic nanocarriers that are relatively uniform in size or shape, such that each synthetic nanocarrier has similar properties. For example, based on the total number of synthetic nanocarriers, at least 80%, at least 90%, or at least 95% of the synthetic nanocarriers may have a minimum or maximum dimension that falls within 5%, 10%, or 20% of the average diameter or average dimension of the synthetic nanocarriers.

[0067] Synthetic nanocarriers can be solid or hollow and can include one or more layers. In some embodiments, each layer has a unique composition and unique properties compared to the other layer(s). By way of example only, synthetic nanocarriers can have a core / shell structure, where the core is one layer (e.g., a polymer core) and the shell is a second layer (e.g., a lipid bilayer or monolayer). Synthetic nanocarriers can include multiple different layers.

[0068] In some embodiments, synthetic nanocarriers may optionally include one or more lipids. In some embodiments, synthetic nanocarriers may include liposomes. In some embodiments, synthetic nanocarriers may include lipid bilayers. In some embodiments, synthetic nanocarriers may include lipid monolayers. In some embodiments, synthetic nanocarriers may include micelles. In some embodiments, synthetic nanocarriers may include a core comprising a polymer matrix surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.). In some embodiments, synthetic nanocarriers may include a non-polymeric core (e.g., a metal particle, a quantum dot, a ceramic particle, a bone particle, a virus particle, a protein, a nucleic acid, a carbohydrate, etc.) surrounded by a lipid layer (e.g., a lipid bilayer, a lipid monolayer, etc.).

[0069] In other embodiments, synthetic nanocarriers may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0070] In some embodiments, synthetic nanocarriers may optionally include one or more amphiphilic entities. In some embodiments, amphiphilic entities may facilitate the production of synthetic nanocarriers with increased stability, improved uniformity, or increased viscosity. In some embodiments, amphiphilic entities may be associated with the inner surface of a lipid membrane (e.g., lipid bilayer, lipid monolayer, etc.). Many amphiphilic entities known in the art are suitable for use in making synthetic nanocarriers in accordance with the present invention.Such amphiphilic entities include, but are not limited to, phosphoglycerides; phosphatidylcholine; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidylethanolamine (DOPE); dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine; cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols such as polyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; surfactant fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides; sorbitan trioleate (Span® 85); glycocholate; sorbitan monolaurate (Span® 20); polysorbate 20 (Tween® 20); polysorbate 60 (Tween® 60); polysorbate 65 (Tween® 100); (Tween® 65); Polysorbate 80 (Tween® 80); Polysorbate 85 (Tween® 85); Polyoxyethylene monostearate; Surfactin; Poloxamer; Sorbitan fatty acid esters such as sorbitan trioleate; Lecithin; Lysolecithin; Phosphatidylserine; Phosphatidylinositol; Sphingomyelin; Phosphatidylethanolamine (cephalin); Cardiolipin; Phosphatidic acid; Cerebroside; Dicetyl phosphate; Dipalmitoyl phosphatase These include phatidylglycerol, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, tyloxapol, poly(ethylene glycol) 5000-phosphatidylethanolamine, poly(ethylene glycol) 400-monostearate, phospholipids, synthetic and / or natural detergents with high surfactant properties, deoxycholate, cyclodextrin, chaotropic salts, ion pairing agents, and combinations thereof. The amphiphilic entity may be a mixture of different amphiphilic entities.Those skilled in the art will appreciate that this is an exemplary list of substances with surfactant effects and is not intended to be comprehensive. Any amphiphilic entity may be used in the production of synthetic nanocarriers to be used in accordance with the present invention.

[0071] In some embodiments, the synthetic nanocarriers may optionally include one or more carbohydrates. The carbohydrates may be natural or synthetic. The carbohydrates may be derivatized natural carbohydrates. In some embodiments, the carbohydrates include monosaccharides or disaccharides, including but not limited to glucose, fructose, galactose, ribose, lactose, sucrose, maltose, trehalose, cellobiose, mannose, xylose, arabinose, glucuronic acid, galacturonic acid, mannuronic acid, glucosamine, galactosamine, and neuraminic acid. In some embodiments, the carbohydrate is a polysaccharide, including, but not limited to, pullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose (HPMC), hydroxycellulose (HC), methylcellulose (MC), dextran, cyclodextran, glycogen, hydroxyethyl starch, carrageenan, glycon, amylose, chitosan, N,O-carboxymethyl chitosan, alginic acid and alginic acid, starch, chitin, inulin, konjac, glucomannan, pustulan, heparin, hyaluronic acid, curdlan, and xanthan. In some embodiments, the synthetic nanocarrier does not include (nor specifically excludes) carbohydrates such as polysaccharides. In some embodiments, the carbohydrate may include carbohydrate derivatives such as sugar alcohols, including, but not limited to, mannitol, sorbitol, xylitol, erythritol, maltitol, and lactitol.

[0072] In some embodiments, the synthetic nanocarriers may include one or more polymers. In some embodiments, the synthetic nanocarriers include one or more polymers that are non-methoxy-terminated Pluronic polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (weight / weight) of the polymers making up the synthetic nanocarriers are non-methoxy-terminated Pluronic polymers. In some embodiments, all of the polymers making up the synthetic nanocarriers are non-methoxy-terminated Pluronic polymers. In some embodiments, the synthetic nanocarriers include one or more polymers that are non-methoxy-terminated polymers. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 99% (weight / weight) of the polymers making up the synthetic nanocarrier are non-methoxy terminated polymers. In some embodiments, all of the polymers making up the synthetic nanocarrier are non-methoxy terminated polymers. In some embodiments, the synthetic nanocarrier comprises one or more polymers that do not include a Pluronic polymer. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 99% (weight / weight) of the polymers making up the synthetic nanocarrier do not contain Pluronic polymers. In some embodiments, all of the polymers making up the synthetic nanocarrier do not contain Pluronic polymers. In some embodiments, such polymers may be surrounded by a coating layer (e.g., liposomes, lipid monolayers, micelles, etc.). In some embodiments, various elements of the synthetic nanocarrier may be attached to the polymer.

[0073] The immunosuppressant may be attached to the synthetic nanocarrier by any of a number of methods. In general, the attachment may be the result of a bond between the immunosuppressant and the synthetic nanocarrier. This bond may result in the immunosuppressant being attached to the surface of the synthetic nanocarrier and / or being contained (encapsulated) within the synthetic nanocarrier. However, in some embodiments, the immunosuppressant is encapsulated by the synthetic nanocarrier as a result of the structure of the synthetic nanocarrier rather than being attached to the synthetic nanocarrier. In a preferred embodiment, the synthetic nanocarrier comprises a polymer as provided herein and the immunosuppressant is attached to the polymer.

[0074] When the attachment occurs as a result of binding between the immunosuppressant drug and the synthetic nanocarrier, the attachment may occur via a coupling moiety. The coupling moiety can be any moiety through which the immunosuppressant drug is bound to the synthetic nanocarrier. Such moieties include covalent bonds, such as amide or ester bonds, as well as another molecule that binds (covalently or non-covalently) the immunosuppressant drug to the synthetic nanocarrier. Such molecules include linkers or polymers, or units thereof. For example, the coupling moiety can include a charged polymer to which the immunosuppressant drug is electrostatically bound. As another example, the coupling moiety can include a polymer or unit thereof to which it is covalently bound.

[0075] In preferred embodiments, the synthetic nanocarriers comprise polymers as provided herein. These synthetic nanocarriers can be entirely polymeric or can be a mix of polymers and other materials.

[0076] In some embodiments, the polymers of the synthetic nanocarriers are linked together to form a polymer matrix. In some of these embodiments, a component, such as an immunosuppressant drug, can be covalently linked to one or more polymers of the polymer matrix. In some embodiments, the covalent linkage is mediated by a linker. In some embodiments, a component can be non-covalently linked to one or more polymers of the polymer matrix. For example, in some embodiments, a component can be encapsulated within, surrounded by, and / or dispersed throughout the polymer matrix. Alternatively or in addition, a component can be linked to one or more polymers of the polymer matrix by hydrophobic interactions, charge interactions, van der Waals forces, and the like. A wide variety of polymers and methods for forming polymer matrices therefrom are known in the art.

[0077] The polymer may be a natural or non-natural (synthetic) polymer. The polymer may be a homopolymer or a copolymer comprising two or more monomers. In terms of sequence, the copolymer may be random, block, or may contain a combination of random and block sequences. Typically, the polymer according to the present invention is an organic polymer.

[0078] In some embodiments, the polymer comprises a polyester, a polycarbonate, a polyamide, or a polyether, or a unit thereof. In other embodiments, the polymer comprises a poly(ethylene glycol) (PEG), a polypropylene glycol, a poly(lactic acid), a poly(glycolic acid), a poly(lactic-co-glycolic acid) copolymer, or a polycaprolactone, or a unit thereof. In some embodiments, the polymer is preferably biodegradable. Thus, in these embodiments, when the polymer comprises a polyether, such as poly(ethylene glycol) or polypropylene glycol, or a unit thereof, the polymer preferably comprises a block copolymer of a polyether and a biodegradable polymer, such that the polymer is biodegradable. In other embodiments, the polymer does not comprise a polyether, such as poly(ethylene glycol) or polypropylene glycol, or a unit thereof, alone.

[0079] Other examples of polymers suitable for use in the present invention include, but are not limited to, polyethylene, polycarbonates (e.g., poly(1,3-dioxane-2-one)), polyanhydrides (e.g., poly(sebacic anhydride)), polypropyl fumarates, polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters (e.g., polylactic acid, polyglycolide, polylactide-glycolide copolymers, polycaprolactones, polyhydroxy acids (e.g., poly(β-hydroxyalkanoates)), poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polyureas, polystyrenes and polyamines, polylysine, polylysine-PEG copolymers, and poly(ethyleneimine), poly(ethyleneimine)-PEG copolymers.

[0080] In some embodiments, polymers according to the invention include polymers approved by the U.S. Food and Drug Administration (FDA) for use in humans under 21 C.FR §177.2600, including, but not limited to, polyesters (e.g., polylactic acid, poly(lactic-co-glycolic acid) copolymers, polycaprolactone, polyvalerolactone, poly(1,3-dioxan-2-one)); polyanhydrides (e.g., poly(sebacic anhydride)); polyethers (e.g., polyethylene glycol); polyurethanes; polymethacrylates; polyacrylates; and polycyanoacrylates.

[0081] In some embodiments, the polymer can be hydrophilic. For example, the polymer can include anionic groups (e.g., phosphate, sulfate, carboxylate); cationic groups (e.g., quaternary amine); or polar groups (e.g., hydroxyl, thiol, amine). In some embodiments, synthetic nanocarriers that include a hydrophilic polymer matrix create a hydrophilic environment within the synthetic nanocarrier. In some embodiments, the polymer can be hydrophobic. In some embodiments, synthetic nanocarriers that include a hydrophobic polymer matrix create a hydrophobic environment within the synthetic nanocarrier. The selection of the hydrophilic or hydrophobic nature of the polymer can have an impact on the properties of the materials that are incorporated (e.g., attached) within the synthetic nanocarrier.

[0082] In some embodiments, the polymer may be modified with one or more moieties and / or functional groups. A variety of moieties or functional groups may be used according to the present invention. In some embodiments, the polymer may be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). Some embodiments may be made using the general teachings of U.S. Patent No. 5,543,158 to Gref et al. or WO publication WO2009 / 051837 by Von Andrian et al.

[0083] In some embodiments, the polymer may be modified with lipid or fatty acid groups. In some embodiments, the fatty acid groups may be one or more of butyric acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, or lignoceric acid. In some embodiments, the fatty acid groups may be one or more of palmitoleic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, arachidonic acid, gadoleic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, or erucic acid.

[0084] In some embodiments, the polymer may be a polyester, including copolymers containing lactic acid and glycolic acid units, such as poly(lactic acid-co-glycolic acid) and poly(lactide-co-glycolide) (collectively referred to herein as "PLGA"); as well as homopolymers containing glycolic acid units (collectively referred to herein as "PGA"), and homopolymers containing poly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide, poly-D-lactide, and poly-D,L-lactide lactic acid units (collectively referred to herein as "PLA"). In some embodiments, exemplary polyesters include, for example, polyhydroxy acids; PEG copolymers, and copolymers of lactide and glycolide (e.g., PLA-PEG copolymer, PGA-PEG copolymer, PLGA-PEG copolymer), and derivatives thereof. In some embodiments, polyesters include, for example, poly(caprolactone), poly(caprolactone)-PEG copolymers, poly(L-lactide-L-lysine) copolymers, poly(serine esters), poly(4-hydroxy-L-proline esters), poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

[0085] In some embodiments, the polymer may be PLGA. PLGA is a biocompatible and biodegradable copolymer of lactic acid and glycolic acid, and the various forms of PLGA are characterized by the ratio of lactic acid:glycolic acid. Lactic acid may be L-lactic acid, D-lactic acid, or D,L-lactic acid. The degradation rate of PLGA can be adjusted by changing the ratio of lactic acid:glycolic acid. In some embodiments, the PLGA to be used according to the present invention is characterized by the ratio of lactic acid:glycolic acid of approximately 85:15, approximately 75:25, approximately 60:40, approximately 50:50, approximately 40:60, approximately 25:75, or approximately 15:85.

[0086] In some embodiments, the polymer may be one or more acrylic acid polymers.In some embodiments, the acrylic acid polymers include, for example, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamide copolymers, poly(methyl methacrylate), poly(methacrylic anhydride), methyl methacrylate, polymethacrylate, poly(methyl methacrylate) copolymers, polyacrylamide, aminoalkyl methacrylate copolymers, glycidyl methacrylate copolymers, polycyanoacrylate, and combinations comprising one or more of the aforementioned polymers.The acrylic acid polymers may include fully polymerized copolymers of acrylic acid and methacrylic acid esters with low content of quaternary ammonium groups.

[0087] In some embodiments, the polymer can be a cationic polymer. Generally, cationic polymers can condense and / or protect the negatively charged strands of nucleic acids. Amine-containing polymers such as poly(lysine) (Zauner et al., 1998, Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, Bioconjugate Chem., 6:7), poly(ethyleneimine) (PEI; Boussif et al., 1995, Proc. Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers (Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897; Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993, Bioconjugate Chem., 4:372) are positively charged at physiological pH and form ion pairs with nucleic acids. In embodiments, synthetic nanocarriers may not include (or may exclude) cationic polymers.

[0088] In some embodiments, the polymer can be a degradable polyester with cationic side chains (Putnam et al., 1999, Macromolecules, 32:3658; Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989, Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633; and Zhou et al., 1990, Macromolecules, 23:3399). Examples of these polyesters include poly(L-lactide-L-lysine) copolymers (Barrera et al., 1993, J. Am. Chem. Soc., 115:11010), poly(serine esters) (Zhou et al., 1990, Macromolecules, 23:3399), poly(4-hydroxy-L-proline esters) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633), and poly(4-hydroxy-L-proline esters) (Putnam et al., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633).

[0089] The properties of and methods for preparing these and other polymers are well known in the art (e.g., U.S. Pat. Nos. 6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148; 5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665; 5,019,379; 5,010,167; 4,806,621; 4,638,045; and 4,946,929; Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2002, J. Am. Chem. Soc., 123:9480; (see, e.g., Uhrich et al., 2001, J. Am. Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer, 1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev., 99:3181). More generally, various methods for synthesizing certain suitable polymers are described in Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press, 1980; Principles of Polymerization by Odian, John Wiley & Sons, Fourth Edition, 2004; Contemporary Polymer Chemistry by Allcock et al., Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and U.S. Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

[0090] In some embodiments, the polymers may be linear or branched polymers. In some embodiments, the polymers may be dendrimers. In some embodiments, the polymers may be substantially crosslinked to each other. In some embodiments, the polymers may be substantially free of crosslinking. In some embodiments, the polymers may be used in accordance with the present invention without undergoing a crosslinking step. It should further be understood that the synthetic nanocarriers may include block copolymers, graft copolymers, blends, mixtures, and / or adducts of any of the aforementioned polymers with other polymers. Those skilled in the art will recognize that the polymers listed herein are representative, but not comprehensive, of the list of exemplary polymers that may be used in accordance with the present invention.

[0091] In some embodiments, synthetic nanocarriers do not include polymeric components. In some embodiments, synthetic nanocarriers may include metal particles, quantum dots, ceramic particles, etc. In some embodiments, non-polymeric synthetic nanocarriers are aggregates of non-polymeric components, such as aggregates of metal atoms (e.g., gold atoms).

[0092] The dosage or dosage form according to the present invention may include pharma- ceutically acceptable excipients, such as preservatives, buffers, saline, or phosphate-buffered saline. The composition may be prepared using conventional pharmaceutical manufacturing and compounding techniques to arrive at a useful dosage form. In some embodiments, the composition of the dosage is suspended in a sterile saline solution for injection together with a preservative. In some embodiments, the synthetic nanocarriers are suspended in a sterile saline solution for injection together with a preservative.

[0093] In embodiments, when preparing synthetic nanocarriers for use with immunosuppressants, methods for attaching components to synthetic nanocarriers may be useful. When the components are small molecules, it may be advantageous to attach the components to a polymer prior to assembly of the synthetic nanocarrier. In embodiments, rather than attaching the components to a polymer with surface groups used to attach the components to the synthetic nanocarrier, it may also be advantageous to prepare the synthetic nanocarrier through the use of these surface groups, and then use this polymer conjugate in the construction of the synthetic nanocarrier.

[0094] In some embodiments, the attachment may use a covalent linker. In embodiments, the components according to the present invention may be covalently attached to the external surface via a 1,2,3-triazole linker formed by 1,3-dipolar cycloaddition of an azide group on the nanocarrier surface with a component containing an alkyne group, or by 1,3-dipolar cycloaddition of an alkyne on the nanocarrier surface with a component containing an azide group. Such cycloaddition is preferably carried out in the presence of a Cu(I) catalyst, together with a suitable Cu(I)-ligand and a reducing agent for reducing the Cu(II) compound to a catalytically active Cu(I) compound. This Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is also referred to as a click reaction.

[0095] Additionally, the covalent attachment may involve a covalent linker, including an amide linker, a disulfide linker, a thioether linker, a hydrazone linker, a hydrazide linker, an imine or oxime linker, a urea or thiourea linker, an amidine linker, an amine linker, and a sulfonamide linker.

[0096] Amide linkers are used to link an amine on one component, such as an immunosuppressant. and , a carboxylic acid group of a second component, such as a nanocarrier BetweenThe amide bond in the linker can be made using any of the conventional amide bond forming reactions with a suitably protected amino acid, such as an N-hydroxysuccinimide activated ester.

[0097] Disulfide linkers are illustratively created through the formation of a disulfide (SS) bond between two sulfur atoms, of the form R1-SS-R2. Disulfide bonds can be formed by thiol exchange of a component containing a thiol / mercaptan group (-SH) with another activated thiol group on a polymer or nanocarrier, or of a nanocarrier containing a thiol / mercaptan group with a component containing an activated thiol group.

[0098] Triazole linkers, specifically the form [ka] 1,2,3-triazoles represented by the formula (wherein R1 and R2 are any chemical entity) are made by 1,3-dipolar cycloaddition of an azide attached to a first component, such as a nanocarrier, with a terminal alkyne attached to a second component, such as an immunosuppressant. The 1,3-dipolar cycloaddition is carried out with or without a catalyst, preferably a Cu(I) catalyst, linking the two components through the 1,2,3-triazole functionality. This chemistry is described in detail in Sharpless et al., Angew. Chem. Int. Ed. 41(14), 2596, (2002) and Meldal, et al., Chem. Rev., 2008, 108(8), 2952-3015, and is often referred to as the "click" reaction or CuAAC.

[0099] In an embodiment, a polymer containing an azide or alkyne group at the end of the polymer chain is prepared. This polymer is then used to prepare a synthetic nanocarrier in such a manner that multiple alkyne or azide groups are located on the surface of the nanocarrier. Alternatively, a synthetic nanocarrier can be prepared by another route and then functionalized with an alkyne or azide group. A building block is prepared in the presence of either an alkyne (if the polymer contains an azide) or an azide (if the polymer contains an alkyne) group. The building block can then be reacted with the nanocarrier with or without a catalyst to covalently attach the building block to the particle through a 1,3-dipolar cycloaddition reaction through a 1,4-disubstituted 1,2,3-triazole linker.

[0100] Thioether linkers are made by the formation of a sulfur-carbon (thioether) bond, illustratively of the form R1-S-R2. Thioethers can be made by either alkylation of a thiol / mercaptan (-SH) group on one component with an alkylating group such as a halide or epoxide on a second component. Thioether linkers can also be formed by Michael addition of a thiol / mercaptan group on one component to an electron-deficient alkene group on a second component containing a maleimide or vinyl sulfone group as a Michael acceptor. In another form, thioether linkers can be prepared by radical thiol-ene reaction of a thiol / mercaptan group on one component with an alkene group on a second component.

[0101] Hydrazone linkers are made by reaction of a hydrazide group on one component with an aldehyde / ketone group on a second component. Hydrazide linkers are formed by the reaction of a hydrazine group on one component with a carboxylic acid group on a second component. Such reactions are generally carried out using chemistry similar to the formation of amide bonds, where the carboxylic acid is activated with an activating reagent.

[0102] Imine or oxime linkers are formed by reaction of an amine or N-alkoxyamine (or aminooxy) group on one component with an aldehyde or ketone group on a second component. Urea or thiourea linkers are prepared by reaction of an amine group on one component with an isocyanate or thioisocyanate group on a second component.

[0103] Amidine linkers are prepared by reaction of an amine group on one component with an imidoester group on a second component. Amine linkers are made by alkylation reaction of an amine group on one component with an alkylating group such as a halide, epoxide, or sulfonate ester group on a second component. Alternatively, amine linkers can also be made by reductive amination of an amine group on one component with an aldehyde or ketone group on a second component with a suitable reducing reagent such as sodium cyanoborohydride or sodium triacetoxyborohydride.

[0104] Sulfonamide linkers are prepared by reaction of an amine group on one component with a sulfonyl halide (such as a sulfonyl chloride) group on a second component. The sulfone linker is made by Michael addition of a nucleophile to a vinyl sulfone. Either the vinyl sulfone or the nucleophile can be on the surface of the nanocarrier or attached to a component.

[0105] The component, preferably the immunosuppressant drug, can also be conjugated to the nanocarrier via non-covalent conjugation methods. For example, a negatively charged immunosuppressant drug can be conjugated to a positively charged nanocarrier through electrostatic adsorption. A component containing a metal ligand can also be conjugated to a nanocarrier containing a metal complex via a metal-ligand complex.

[0106] In embodiments, the building blocks can be attached to a polymer, such as polylactide-block-polyethylene glycol, prior to assembly of the synthetic nanocarrier, or the synthetic nanocarrier can be formed with reactive or activatable groups on its surface. In the latter case, the building blocks can be prepared with groups that match the attachment chemistry presented by the surface of the synthetic nanocarrier. In other embodiments, the peptide building blocks can be attached to the VLP or liposome using a suitable linker. A linker is a compound or reagent that can attach two molecules to each other. In one embodiment, the linker can be a homobifunctional or heterobifunctional reagent as described in Hermanson 2008. For example, a VLP or liposomal synthetic nanocarrier containing carboxylic acid groups on its surface can be treated with the homobifunctional linker adipic acid dihydrazide (ADH) in the presence of EDC to form the corresponding synthetic nanocarrier with an ADH linker. The resulting ADH-linked synthetic nanocarriers are then conjugated with a peptide component containing an acid group via the other end of the ADH linker on the nanocarrier to produce the corresponding VLP or liposome-peptide conjugate.

[0107] For a detailed description of available conjugation methods, see Hermanson GT "Bioconjugate Techniques", 2nd Edition Published by Academic Press, Inc., 2008. In addition to covalent attachment, components can be attached by adsorption to preformed synthetic nanocarriers or by encapsulation during formation of the synthetic nanocarriers.

[0108] Any immunosuppressant drug can be used to attach to the synthetic nanocarrier as provided herein. Immunosuppressants include, but are not limited to, statins; mTOR inhibitors such as rapamycin or rapamycin analogs; TGF-β signaling agents; TGF-β receptor agonists; histone deacetylase (HDAC) inhibitors; corticosteroids; inhibitors of mitochondrial function such as rotenone; P38 inhibitors; NF-κβ inhibitors; adenosine receptor agonists; prostaglandin E2 agonists; phosphodiesterase inhibitors such as phosphodiesterase 4 inhibitors; Proteasome inhibitors, kinase inhibitors, G protein-coupled receptor agonists, G protein-coupled receptor antagonists, glucocorticoids, retinoids, cytokine inhibitors, cytokine receptor inhibitors, cytokine receptor activators, peroxisome proliferator-activated receptor antagonists, peroxisome proliferator-activated receptor agonists, histone deacetylase inhibitors, calcineurin inhibitors, phosphatase inhibitors and oxidized ATP. Immunosuppressants also include IDO, vitamin D3, cyclosporin A, aryl hydrocarbon receptor inhibitors, resveratrol, azathioprine, 6-mercaptopurine, aspirin, niflumic acid, estriol, tripolide, interleukins (e.g., IL-1, IL-10), cyclosporin A, SIRNAs targeting cytokines or cytokine receptors, etc.

[0109] Examples of statins include atorvastatin (LIPITOR®, TORVAST®), cerivastatin, fluvastatin (LESCOL®, LESCOL® XL), lovastatin (MEVACOR®, ALTOCOR®, ALTOPREV®), mevastatin (COMPACTIN®), pitavastatin (LIVALO®, PIAVA®), rosuvastatin (PRAVACHOL®, SELEKTINE®, LIPOSTAT®), rosuvastatin (CRESTOR®), and simvastatin (ZOCOR®, LIPEX®).

[0110] Examples of mTOR inhibitors include rapamycin and its analogues (e.g., CCL-779, RAD001, AP23573, C20-methallylrapamycin (C20-Marap), C16-(S)-butylsulfonamidorapamycin (C16-BSrap), C16-(S)-3-methylindolerapamycin (C16-iRap) (Bayle et al. Chemistry & Biology 2006,13:99-107), AZD8055, BEZ235 (NVP-BEZ235), chrysophanic acid (chrysophanol), deforolimus (MK-8669), everolimus (RAD0001), KU-0063794, PI-103, PP242, temsirolimus, and WYE-354 (available from Selleck, Houston, TX, USA).

[0111] Examples of TGF-β signaling agents include TGF-β ligands (e.g., activin A, GDF1, GDF11, bone morphogenetic protein, nodal, TGF-β) and their receptors (e.g., ACVR1B, ACVR1C, ACVR2A, ACVR2B, BMPR2, BMPR1A, BMPR1B, TGFβRI, TGFβRII), R-SMADS / CO-SMADS (e.g., SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD8), and ligand inhibitors (e.g., follistatin, noggin, chordin, DAN, lefty, LTBP1, THBS1, decorin).

[0112] Examples of inhibitors of mitochondrial function include atractyloside (dipotassium salt), bongkrekic acid (triammonium salt), carbonyl cyanide M-chlorophenylhydrazone, carboxyatractyloside (e.g., from Atractylis gummifera), CGP-37157, (-)-deguelin (e.g., from Mundulea sericea), F16, hexokinase II VDAC binding domain peptide, oligomycin, rotenone, Ru360, SFK1, and valinomycin (e.g., from Streptomyces fulvissimus) (EMD4Biosciences, USA).

[0113] Examples of P38 inhibitors include SB-203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole), SB-239063 (trans-1-(4 hydroxycyclohexyl)-4-(fluorophenyl)-5-(2-methoxy-pyrimidin-4-yl)imidazole), SB-220025 (5-(2 amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinyl)imidazole)), and ARRY-797.

[0114] Examples of NF (e.g., NK-κβ) inhibitors include IFRD1, 2-(1,8-naphthyridin-2-yl)-phenol, 5-aminosalicylic acid, BAY 11-7082, BAY 11-7085, CAPE (caffeic acid phenethyl ester), diethyl maleate, IKK-2 inhibitor IV, IMD 0354, lactacystin, MG-132 [Z-Leu-Leu-Leu-CHO], NFκB activation inhibitor III, NF-κB activation inhibitor II, JSH-23, parthenolide, phenylarsine oxide (PAO), PPM-18, pyrrolidine dithiocarbamic acid ammonium salt, QNZ, RO 106-9920, rocaglamide, rocaglamide AL, rocaglamide C, rocaglamide I, rocaglamide J, rocaglaol, (R)-MG-132, sodium salicylate, triptolide (PG490), and wedelolactone.

[0115] Examples of adenosine receptor agonists include CGS-21680 and ATL-146E. Examples of prostaglandin E2 agonists include E-prostanoid 2 and E-prostanoid 4.

[0116] Examples of phosphodiesterase inhibitors (non-selective and selective inhibitors) are caffeine, aminophylline, IBMX (3-isobutyl-1-methylxanthine), paraxanthine, pentoxifylline, theobromine, theophylline, methylated xanthines, vinpocetine, EHNA (erythro-9-(2-hydroxy-3-nonyl)adenine), anagrelide, enoximone (PERFAN®), milrinone, levosimendan, mesembrine, ibudilast, These include piclamilast, luteolin, drotaverine, roflumilast (DAXAS™, DALIRESP™), sildenafil (REVATION®, VIAGRA®), tadalafil (ADCIRCA®, CIALIS®), vardenafil (LEVITRA®, STAXYN®), udenafil, avanafil, icariin, 4-methylpiperazine, and pyrazolopyrimidine-7-one.

[0117] Examples of proteasome inhibitors include bortezomib, disulfiram, epigallocatechin-3-gallate, and salinosporamide A. Examples of kinase inhibitors include bevacizumab, BIBW 2992, cetuximab (Erbitux®), imatinib (GLEEVEC®), trastuzumab (Herceptin®), gefitinib (IRESSA®), ranibizumab (LUCENTIS®), pegaptanib, sorafenib, dasatinib, sunitinib, erlotinib, nilotinib, lapatinib, panitumumab, vandetanib, E7080, pazopanib, and mubritinib.

[0118] Examples of glucocorticoids include hydrocortisone (cortisol), cortisone acetate, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate (DOCA), and aldosterone.

[0119] Examples of retinoids include retinol, retinal, tretinoin (retinoic acid, RETIN-A®), isotretinoin (ACCUTANE®, AMNESTEEM®, CLARAVIS®, SOTRET®), alitretinoin (PANRETIN®), etretinate (TEGISON™) and its metabolite acitretin (SORIATANE®), tazarotene (TAZORAC®, AVAGE®, ZORAC®), bexarotene (TARGRETIN®), and adapalene (DIFFERIN®).

[0120] Examples of cytokine inhibitors include IL1ra, IL1 receptor antagonist, IGFBP, TNF-BF, uromodulin, alpha-2-macroglobulin, cyclosporine A, pentamidine, and pentoxifylline (PENTOPAK®, PENTOXIL®, TRENTAL®). Examples of peroxisome proliferator-activated receptor antagonists include GW9662, PPARγ antagonist III, G335, and T0070907 (EMD4 Biosciences, USA).

[0121] Examples of peroxisome proliferator-activated receptor agonists include pioglitazone, ciglitazone, clofibrate, GW1929, GW7647, L-165,041, LY 171883, PPARγ activators, Fmoc-Leu, troglitazone, and WY-14643 (EMD4Biosciences, USA).

[0122] Examples of histone deacetylase inhibitors include hydroxamic acids (or hydroxamates) such as trichostatin A, cyclic tetrapeptides (such as trapoxin B) and depsipeptides, aliphatic acid compounds such as benzamides, electrophilic ketones, phenylbutyrates, and valproic acid, hydroxamic acids such as vorinostat (SAHA), belinostat (PXD101), LAQ824, and panobinostat (LBH589), benzamides such as entinostat (MS-275), CI994, and mocetinostat (MGCD0103), nicotinamides, derivatives of NAD, dihydrocoumarins, naphthopyranones, and 2-hydroxynaphthaldehydes.

[0123] Examples of calcineurin inhibitors include cyclosporine, pimecrolimus, voclosporin, and tacrolimus. Examples of phosphatase inhibitors include BN82002 hydrochloride, CP-91149, calyculin A, cantharidic acid, cantharidin, cypermethrin, ethyl-3,4-defostatin, fostriecin sodium salt, MAZ51, methyl-3,4-defostatin, NSC 95397, norcantharidin, okadaic acid ammonium salt from prorocentrum concavum, okadaic acid, okadaic acid potassium salt, okadaic acid sodium salt, phenylarsine oxide, various phosphatase inhibitor cocktails, protein phosphatase 1C, protein phosphatase 2A inhibitor protein, protein phosphatase 2A1, protein phosphatase 2A2, and sodium orthovanadate.

[0124] D. Methods of Making and Using the Compositions and Related Methods Viral vectors can be produced by methods known to those skilled in the art or as otherwise described herein.For example, viral vectors can be constructed and / or purified using the methods described in, for example, U.S. Patent No. 4,797,368 and Laughlin et al., Gene, 23, 65-73 (1983).

[0125] Viral vectors such as AAV vectors may be produced using recombinant methods. For example, the methods may involve culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or a fragment thereof; a functional rep gene; a recombinant AAV vector made of an AAV inverted terminal repeat (ITR) and a transgene; and sufficient helper functions to allow packaging of the recombinant AAV vector into the AAV capsid protein.

[0126] The components to be cultured in the host cell to package the viral vector into a capsid may be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., recombinant viral vector, rep sequence, cap sequence, and / or helper functions) may be provided by a stable host cell engineered to contain one or more of the required components using methods known to those skilled in the art. Most preferably, such a stable host cell may contain the required component(s) under the control of an inducible promoter. However, the required component(s) may be under the control of a constitutive promoter. The recombinant viral vector, rep sequence, cap sequence, and helper functions to produce the viral vector may be delivered to the packaging host cell using any suitable genetic element. The selected genetic element may be delivered by any suitable method, including the methods described herein. The methods used to construct any embodiment of the present invention are known to those skilled in nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al., J. Virol., 70:520-532 (1993) and U.S. Patent No. 5,478,745.

[0127] In some embodiments, recombinant AAV vectors may be produced using a triple transfection method (e.g., as described in detail in U.S. Pat. No. 6,001,650, the contents of which regarding triple transfection method are incorporated herein by reference). Typically, recombinant AAV is produced by transfecting a host cell with a recombinant AAV vector (such as one containing a transgene) to be packaged into an AAV particle, an AAV helper function vector, and an auxiliary function vector. In general, the AAV helper function vector encodes AAV helper function sequences (rep and cap) that function in trans for replication and encapsulation of propagating AAV. Preferably, the AAV helper function vector supports efficient AAV vector production without producing any detectable wild-type AAV virions (i.e., AAV virions containing functional rep and cap genes). The auxiliary function vector may encode nucleotide sequences for non-AAV-derived viral and / or cellular functions that AAV depends on for replication. The accessory functions include those functions required for AAV replication, including but not limited to those parts involved in AAV gene transcription, stage-specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression product, and activation of AAV capsid ad assembly. The virus-based accessory functions can be derived from any of the known helper viruses, such as adenovirus, herpesvirus (other than herpes simplex virus type 1), and vaccinia virus. Other methods for producing viral vectors are known in the art. Moreover, viral vectors are commercially available.

[0128] With respect to synthetic nanocarriers attached to immunosuppressant drugs, methods for attaching components to synthetic nanocarriers may be useful. Synthetic nanocarriers may be prepared using a wide variety of methods known in the art. For example, synthetic nanocarriers may be formed by methods such as nanoprecipitation, flow focusing using fluidic channels, spray drying, single and double emulsion solvent evaporation, solvent extraction, phase separation, milling, microemulsion procedures, microfabrication, nanofabrication, sacrificial layers, simple and complex coacervation, and other methods known to those skilled in the art. Alternatively or in addition, aqueous and organic solvent syntheses of monodisperse semiconductor, conductive, magnetic, organic, and other nanomaterials have been described (Pellegrino et al., 2005, Small, 1:48; Murray et al., 2000, Ann. Rev. Mat. Sci., 30:545; and Trindade et al., 2001, Chem. Mat., 13:3843). Additional methods have been described in the literature (see, e.g., Doubrow, Ed., "Microcapsules and Nanoparticles in Medicine and Pharmacy," CRC Press, Boca Raton, 1992; Mathiowitz et al., 1987, J. Ontrol. Release, 5:13; Mathiowitz et al., 1987, Reactive Polymers, 6:275; and Mathiowitz et al., 1988, J. Appl. Polymer Sci., 35:755; U.S. Patent Nos. 5,578,325 and 6,007,845; P. Paolicelli et al., "Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like Particles" Nanomedicine. 5(6):843-853 (2010)).

[0129] Various materials are described in C. Astete et al., “Synthesis and characterization of PLGA nanoparticles” J. Biomater. Sci. Polymer Edn, Vol. 17, No. 3, pp. 247-289 (2006); K. Avgoustakis “Pegylated Poly(Lactide) and Poly(Lactide-Co-Glycolide) Nanoparticles: Preparation, Properties and Possible Applications in Drug Delivery”Current Drug Delivery 1:321-333(2004);C.Reis et al.,“Nanoencapsulation I. Methods for preparation of drug-loaded polymeric nanoparticles”Nanomedicine 2:8-21(2006);P.Paolicelli et al.,“Surface-modified PLGA-based Nanoparticles that can Efficiently Associate and Deliver Virus-like A variety of methods may be used to encapsulate materials into synthetic nanocarriers as desired, including, but not limited to, those disclosed in U.S. Pat. No. 6,632,671 to Unger, issued Oct. 14, 2003.

[0130] In some embodiments, synthetic nanocarriers are prepared by a nanoprecipitation process or spray drying. The conditions used in preparing the synthetic nanocarriers may be varied to produce particles of desired size or characteristics (e.g., hydrophobicity, hydrophilicity, outer morphology, "stickiness", shape, etc.). The method of preparing the synthetic nanocarriers and the conditions used (e.g., solvent, temperature, concentration, air flow rate, etc.) may depend on the composition of the material and / or polymer matrix to be attached to the synthetic nanocarriers.

[0131] If synthetic nanocarriers prepared by any of the above methods have a size range outside the desired range, such synthetic nanocarriers can be sized, for example, using sieves.

[0132] The elements (i.e., components) of the synthetic nanocarrier may be attached to the synthetic nanocarrier as a whole, for example by one or more covalent bonds, or may be attached using one or more linkers. Additional methods for functionalizing synthetic nanocarriers may be adapted from U.S. Patent Application Publication No. 2006 / 0002852 to Saltzman et al., U.S. Patent Application Publication No. 2009 / 0028910 to DeSimone et al., or International Patent Application Publication No. WO / 2008 / 127532 A1 to Murthy et al.

[0133] Alternatively or in addition, the synthetic nanocarriers can be attached to the components directly or indirectly through non-covalent interactions. In non-covalent embodiments, the non-covalent coupling is mediated by non-covalent interactions including, but not limited to, charge interactions, affinity interactions, metal coordination, physical adsorption, host-host interactions, hydrophobic interactions, TT stacking interactions, hydrogen bonding interactions, van der Waals interactions, magnetic interactions, electrostatic interactions, dipole-dipole interactions, and / or combinations thereof. Such couplings may be arranged to be on the exterior or interior surfaces of the synthetic nanocarriers. In embodiments, encapsulation and / or absorption are forms of coupling.

[0134] The compositions provided herein may contain inorganic or organic buffers (e.g., sodium or potassium salts of phosphate, carbonic acid, acetate, or citric acid), and pH adjusters (e.g., hydrochloric acid, sodium or potassium hydroxide, salts of citric or acetate, amino acids and their salts), antioxidants (e.g., ascorbic acid, alpha-tocopherol), surfactants (e.g., polysorbate 20, polysorbate 80, polyoxyethylene 9-10 nonylphenol, sodium deoxycholate), solutions and / or cryo / freezing agents. (lyo)Stabilizers (e.g., sucrose, lactose, mannitol, trehalose), osmolality adjusting agents (e.g., salts or sugars), antimicrobial agents (e.g., benzoic acid, phenol, gentamicin), antifoaming agents (e.g., polydimethylsiloxane, preservatives (e.g., thimerosal, 2-phenoxyethanol, EDTA), polymeric stabilizers and viscosity adjusting agents (e.g., polyvinylpyrrolidone, poloxamer 488, carboxymethylcellulose), and co-solvents (e.g., glycerol, polyethylene glycol, ethanol).

[0135] The composition according to the present invention may include pharma- ceutically acceptable excipients. The composition may be made using conventional pharmaceutical manufacturing and compounding techniques to reach a useful dosage form. Suitable techniques for use in the practice of the present invention may be found in Handbook of Industrial Mixing: Science and Practice, Edited by Edward L. Paul, Victor A. Atiemo-Obeng, and Suzanne M. Kresta, 2004 John Wiley & Sons, Inc.; and Pharmaceutics: The Science of Dosage Form Design, 2nd Ed. Edited by ME Auten, 2001, Churchill Livingstone. In one embodiment, the composition is in sterile saline solution for injection, together with preservatives.

[0136] The compositions of the invention may be made in any suitable manner, and it should be understood that the invention is in no way limited to compositions that may be produced using the methods described herein. Selection of an appropriate method of manufacture may require careful consideration of the properties of the particular moieties involved.

[0137] In some embodiments, the composition is manufactured under sterile conditions or terminally sterilized. This can ensure that the resulting composition is sterile and non-infectious, thus improving safety when compared with non-sterile compositions. This provides a valuable safety measure, especially when the subject receiving the composition has an immune deficiency, suffers from an infectious disease, and / or is susceptible to infectious diseases. In some embodiments, the composition may be lyophilized and stored in suspension or as lyophilized powder, relying on a formulation strategy that does not lose activity for long periods of time.

[0138] Administration according to the present invention may be by various routes, including but not limited to subcutaneous, intravenous, or intraperitoneal. The compositions referred to herein may be manufactured and prepared using conventional methods for administration, such as combination administration.

[0139] The compositions of the present invention may be administered in an effective amount, such as an effective amount described elsewhere herein. The dosage form may contain varying amounts of immunosuppressant drugs according to the present invention. The dosage form may contain varying amounts of viral vectors according to the present invention. The amount of each component present in the dosage form may vary according to the nature of the component, the therapeutic benefit to be achieved, and other such parameters. In embodiments, dose range studies may be performed to establish the optimal therapeutic amount of the component present in the dosage form. In embodiments, the component is present in the dosage form in an amount effective to reduce an undesired immune (e.g., humoral) response to the viral vector upon administration to a subject, and / or to provide effective and / or durable and / or equivalent expression. It may be possible to determine the amount of the component effective to reduce an undesired immune response in a subject using conventional dose range studies and techniques. The dosage form may be administered at various frequencies (i.e., according to a dosing schedule).

[0140] Another aspect of the present disclosure relates to a kit. In some embodiments, the kit comprises one or more doses of a synthetic nanocarrier attached to one or more doses of a viral vector and / or an immunosuppressant drug as provided herein. Each of the doses of the kit can be contained in a separate container in the kit or in the same container. In some embodiments, the container is a vial or an ampoule. In some embodiments, each of the doses can be contained in a solution separate from the container, so that the doses can be added to the container at a later time. In some embodiments, the doses are each in a lyophilized form, in a separate container or in the same container, so that they can be reconstituted at a later time. In some embodiments, the kit further comprises instructions for reconstitution, mixing, administration, etc. In some embodiments, the instructions include a description of the methods described herein. The instructions can be in any suitable form, for example as a printed insert or label. In some embodiments, the kit further comprises one or more syringes.

[0141] The dosing schedule may be determined by varying the number of doses(es) and / or the length of time between doses(es) and assessing the undesired immune response to the viral vector and / or expression of the transgene or nucleic acid material. For example, after administering a dose(s) of the viral vector and / or a dose(s) of the synthetic nanocarrier attached to the immunosuppressant drug, transgene expression and / or undesired immune response to the viral vector may be measured. This undesired immune response and / or expression may be compared to the same type of immune response and / or expression that occurs without the administration(s) of the synthetic nanocarrier attached to the immunosuppressant drug, such as when the administration of the viral vector occurs without the co-administration of the synthetic nanocarrier attached to the immunosuppressant drug as provided herein. The dosing schedule may be determined by starting with a test schedule and using known scaling techniques (such as allometric or isometric scaling) as needed. In another embodiment, the dosing schedule may be determined by testing various schedules in subjects, for example through direct experimentation based on experience and guiding data.

[0142] example Example 1: Synthesis of synthetic nanocarriers containing immunosuppressant drugs A synthetic nanocarrier (e.g., PLA and / or PLA-PEG nanocarrier) containing (e.g., encapsulating) an immunosuppressant drug, such as rapamycin; (e.g., ImmTOR) was produced. Preferably, in some embodiments of any one of the methods or compositions provided herein, the synthetic nanocarrier containing an immunosuppressant drug is produced by any one of the methods in US Publication No. US 2016 / 0128986 A1 and US Publication No. US 2016 / 0128987 A1, the described methods of such production and the resulting synthetic nanocarrier are incorporated herein by reference in their entirety. In any one of the methods or compositions provided herein, the synthetic nanocarrier containing an immunosuppressant drug, such as rapamycin, is such an incorporated synthetic nanocarrier.

[0143] Example 2: Systemic immunogenicity considerations for adeno-associated virus (AAV)-mediated gene therapy AAV immunogenicity and AAV toxicity represent two major challenges for the gene therapy field and are in many cases closely linked. AAV vector immunogenicity is believed to cause or exacerbate many adverse events associated with AAV gene therapy, such as hepatotoxicity and thrombotic microangiopathy. Moreover, these adverse events tend to correlate with vector dose, increasing both in prevalence and severity at higher doses. Not surprisingly, higher vector doses are also associated with increased immunogenicity, leading to a vicious cycle when vector doses of 1E14 vg / kg or more are required for efficacy in neuromuscular diseases. These issues will likely require a multifaceted approach to improve vector transduction efficiency and mitigate vector immunogenicity, and perhaps a shift in the “one-and-done” paradigm of gene therapy.

[0144] The immunogenicity of AAV gene therapy is much more complex and nuanced than that of most protein biological therapies, which are typically only affected by anti-drug antibodies (ADA). ADA also limits systemic AAV gene therapy through pre-existing anti-AAV neutralizing antibodies that arise through natural exposure to wild-type AAV and limit the patient's eligibility for gene therapy, and through the de novo formation of neutralizing ADA that occurs after treatment and prevents the ability to re-administer AAV gene therapy. However, much of the toxicity of AAV gene therapy appears to be driven by innate immune responses and adaptive CD8 T cell responses.

[0145] Early clinical trials in hemophilia reported delayed elevation of serum levels of liver transaminases, indicative of hepatitis and hepatotoxicity. In some patients, increases in liver enzymes correlated with decreased transgene expression and the appearance of circulating capsid-specific CD8 T cells. In many cases, elevated liver transaminase levels and further loss of transgene expression could be successfully treated with steroids. Induction of adaptive immune responses points to activation of innate antigen-presenting cells, such as dendritic cells or macrophages. APCs express Toll-like receptors (TLRs), which are involved in the recognition of pathogen-associated molecular patterns. The DNA payload of AAV vectors may contain CpG motifs that bind and activate TLR9, an innate immune sensor for unmethylated CpG dinucleotides. Similarly, recognition of the capsid itself by TLR2 can also lead to activation of APCs, which can lead to induction of adaptive immune responses. An interesting aspect of AAV gene therapy-associated hepatotoxicity is its delayed onset, typically 4–8 weeks after administration, suggesting that AAV capsid antigen persists in some tissues and that some secondary event, or possibly cumulative hepatic stress, leads to CD8 T cell activation.

[0146] Adverse events associated with elevated hepatic transaminases are now widely reported in clinical trials of AAV gene therapy, with increasing prevalence at higher vector doses. AAV gene therapy for neuromuscular diseases has typically required doses in excess of 1E14vg / kg. Onasemnogene abeparvovec, the first systemic AAV gene therapy approved by the FDA for AAV9 therapy for spinal muscular atrophy (SMA), has been administered to over 800 patients. Approximately one-third of patients receiving the 1.1E14vg / kg dose experienced at least one adverse event of hepatotoxicity. Three patients were reported to experience severe hepatotoxicity 7-8 weeks after treatment. Liver biopsies indicated hepatocellular degeneration, and inflammatory infiltrates contained primarily CD8 T cells. All three patients recovered after treatment with methylprednisolone. Unfortunately, however, three patients with X-linked myotubular myopathy (XLMTM) died 20–40 weeks after receiving a vector dose of 3E14 vg / kg. The patients were found to have signs of severe hepatotoxicity, with bilirubin levels 35–60 times the upper limit of normal and delayed elevation of AST and ALT, and evidence of cholestasis, periportal and bile ductal reaction, and secondary fibrosis at autopsy. The lack of inflammatory cell infiltration and reports pointing to ineffective treatment with steroids and immunosuppressants suggest that the toxicity was not immune-related. All three patients showed preexisting evidence of hyperbilirubinemia and intrahepatic cholestasis. However, an early innate immune response to the large AAV dose could have contributed to hepatic stress, leading to exacerbation of the underlying disease. The trial was stopped and eventually restarted at a lower dose of 1E14 vg / kg. Unfortunately, a fourth patient died after treatment with the lower dose.

[0147] High vector doses of AAV are also associated with thrombotic microangiopathy (TMA), or atypical hemolytic uremic syndrome (aHUS), a syndrome characterized by hemolytic anemia, thrombocytopenia, and acute kidney injury, and associated with complement activation. Complement is a key component of the innate immune system, composed of plasma proteins that undergo a complex cascade of biochemical reactions that can result in direct killing of pathogens, opsonization for pathogen clearance, and chemoattraction and activation of innate immune cells and release of vasoactive mediators. AAV can activate complement through binding of antibodies that fix complement (classical pathway) or direct binding to complement components (alternative pathway). Three cases of TMA have been reported in SMA. Although over 800 patients have been treated, the overall incidence is low; nevertheless, one affected subject had a complication of persistent hypertension, while another took 3 months to resolve. In DMD, two small clinical trials with two different AAV product candidates reported four cases of severe adverse reactions with aHUS among 15 total patients dosed (Mendell Mol Ther 2021). One case occurred at a dose of 5E13vg / kg, while the other three cases occurred at vector doses of 1-3E14vg / kg. The results of three cases of myocarditis led to an amendment of the phase 3 clinical trial in DMD to exclude patients with certain mutations. Furthermore, unfortunately, a patient death was reported in the phase 1b trial. Further details are not available and the clinical trial has been put on hold.

[0148] The different manifestations and relative incidence of adverse reactions suggest that multiple variables may contribute to the overall risk, such as the vector design, the nature of the vector manufacturing and quality, the underlying disease, and comorbidities. A retrospective analysis of a hemophilia B gene therapy trial suggests that higher CpG content correlated with a higher incidence of liver enzyme elevations, stronger CD8 T cell responses, and lower persistent transgene expression. In a third clinical program, DMD, 77 patients have been treated, and no serious adverse events related to aHUS have yet been reported. The basis for the higher incidence of aHUS in two programs and not in the third is unclear. It is assumed that different capsids will have different profiles with respect to complement fixation or binding to pre-existing antibodies. One of the major unknowns in all AAV clinical trials is the degree of impurities from the manufacturing process, such as the percentage of empty capsids or host cell impurities. Because AAV dose is calculated based on determination of the vector genome (or "full" capsid), the ratio of empty:full capsids can have a profound bearing on total capsid dose.

[0149] Given the notable successes and continuing promise of the AAV field, the potential to improve the safety and efficacy of recombinant AAV vectors remains great. A capsid dose of 1E14vg / kg represents a dose of over one quadrillion viral particles (not including empty capsids) in young children, which is 10-30 times greater than the total number of somatic cells. Increasing the ratio of filled:empty capsids and reducing CpG content provides an immediately actionable strategy to improve safety. Another potential strategy to increase transduction efficiency and reduce CD8 T cell responses is to introduce capsid mutations that minimize proteasomal processing, resulting in capsid degradation and presentation of immunogenic epitopes on MHC class I molecules. Long-term strategies include sophisticated capsid engineering approaches to design or evolve capsids that better target specific tissues, such as muscle. Improved promoter and enhancer design may also improve efficacy at lower doses. In addition, lower vector doses may be possible through engineering of more efficient transgenes, as exemplified by the Padua variant used in hemophilia B. Ultimately, a breakthrough to fundamentally improve transduction efficiency will require a better understanding of the key rate-limiting steps of AAV transduction, including passage through the cell from endosomes to the nucleus, viral uncoating, maintenance as a stable episome, and interaction with host cell factors.

[0150] Both capsid engineering and pharmacological strategies are being pursued to reduce the immunogenicity of AAV capsids. Some strategies, such as mutagenesis of the capsid to remove antibody epitopes, or the use of plasma exchange or IgG proteases to remove anti-AAV antibodies, address only the pre-existing antibody component without addressing either innate immunity or T cell responses. Indeed, the very presence of pre-existing anti-AAV antibodies indicates prior exposure to AAV and a high likelihood that AAV-specific memory T and B cells are present. High vector doses that can overcome low levels of neutralizing antibodies have allowed some groups to relax the threshold antibody titer for inclusion in clinical trials, even though it remains possible that pre-existing memory T cells and complement fixation by pre-existing low-titer antibodies may exacerbate the toxicity observed with high vector doses. For example, combination immunosuppressive regimens of rapamycin and corticosteroids or rapamycin and rituximab are being clinically evaluated for immunogenicity reduction. Rapamycin is an inhibitor of the mTOR pathway, which can be administered chronically to inhibit the activation of effector T cells. Rapamycin encapsulated in nanoparticles (also referred to as ImmTOR in some embodiments) has also been evaluated for the induction of antigen-specific immune tolerance to AAV vectors. A dose of ImmTOR nanoparticles administered during AAV gene therapy treatment has been shown to inhibit T and B cell responses to AAV, allowing for re-administration of the vector. ImmTOR has also been shown to have hepatoprotective properties in animal models of liver inflammation. Of note, rapamycin has also been shown to enhance AAV vector transduction through the induction of autophagy.

[0151] The risks are high to mitigate the toxicity of high vector doses. The benefits of AAV gene therapy are undeniable in SMA and extremely promising in DMD. Those patients who tolerated the treatment, even with XLMTM, showed significant improvement. Further improvements may be achievable if the therapeutic window can be safely widened. The ultimate goal would be to provide similar efficacy at lower vector doses. This may require a combination of vector engineering and pharmacological strategies to increase vector transduction efficiency and reduce immunogenicity. AAV gene therapy has historically been considered a "one and done" treatment, meaning that a single dose of treatment is expected to provide several years, if not a lifetime, of therapeutic benefit. This view has been challenged by the waning efficacy in hemophilia A gene therapy trials and the loss of efficacy observed in patients who experienced liver toxicity. If the barrier to vector re-administration, currently limited by the formation of high-titer neutralizing antibodies after initial exposure, could be overcome, it would not only open the possibility of restoring therapeutic benefit to patients who have experienced loss of transgene expression, but also provide an avenue for the treatment of neuromuscular diseases, etc., by allowing multiple smaller doses of gene therapy rather than a single high dose of 1-3E14 vg / kg. The potential to provide therapeutic outcomes by delivering multiple lower doses over a limited period of time (e.g., 1-2 weeks or 1-3 months) could be the next gene therapy paradigm facilitating the next wave of approved gene therapeutics with lower toxicity profiles for unmet rare diseases - "lower and slower" could be prioritized over "one and done."

Claims

1. (i) one or more doses of a viral vector, such as an AAV vector, not attached to any synthetic nanocarriers, where each dose or doses of the viral vector is a lower dose; and (ii) at least one dose of a synthetic nanocarrier attached to an immunosuppressant drug, such as rapamycin, and not including any viral vector antigen of the viral vector; and administering the same to a patient, comprising administering wherein the lower dose results in transgene expression that is lower than the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug, but equivalent to the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug; Optionally, the method follows an administration schedule that reduces unwanted immune (e.g., humoral) responses to the viral vector and / or results in effective expression of the transgene or nucleic acid material and / or provides persistent expression of the transgene or nucleic acid material and / or results in equivalent expression of the transgene.

2. 2. The method of claim 1, wherein the dosages of (i) and (ii) are administered concomitantly.

3. 3. The method of claim 1 or 2, wherein the dosing comprising (i) and (ii) is repeated, optionally within 70 days, 2 months, or 1 month of the previous dosing of (i) and (ii).

4. 4. The method of any one of claims 1 to 3, wherein the method further comprises assessing unwanted immune responses and / or expression of the introduced gene or nucleic acid material in the subject prior to and / or after the administration(s).

5. The method of any one of claims 1 to 4, wherein the method further comprises identifying the subject as having or at risk of having an unwanted immune response to the viral vector and / or as in need of effective or permanent expression of the transgene or nucleic acid material.

6. (i) more than one dose of a viral vector, such as an AAV vector, not attached to any synthetic nanocarrier, where each dose of viral vector is a lower dose; and / or (ii) at least one dose of a synthetic nanocarrier attached to an immunosuppressant drug, such as rapamycin, and not including any viral vector antigen of the viral vector; A composition comprising: wherein the lower dose results in transgene expression that is lower than the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug, but equivalent to the dose of the viral vector administered without the concomitant administration of the synthetic nanocarrier attached to the immunosuppressant drug; Optionally, the method is for use in a method of reducing an unwanted immune (e.g. humoral) response to the viral vector and / or in a method of providing effective and / or persistent expression of a transgene or nucleic acid material and / or in a method of providing equivalent transgene expression.

7. 7. The method or composition of any one of claims 1 to 6, wherein the method further comprises determining a protocol or administration schedule for (i) and (ii).

8. The method or composition of any one of claims 1 to 7, wherein the method is any of the methods provided herein.

9. The composition of any one of claims 6 to 8, wherein the composition is a kit and the dosages are each contained in a container in the kit.

10. The composition of any one of claims 6 to 9, wherein the composition further comprises a pharma- ceutically acceptable carrier.

11. 11. The method or composition of any one of claims 1 to 10, wherein the immunosuppressant is a statin, an mTOR inhibitor, a TGF-β signaling agent, a corticosteroid, an inhibitor of mitochondrial function, a P38 inhibitor, an NF-κβ inhibitor, an adenosine receptor agonist, a prostaglandin E2 agonist, a phosphodiesterase 4 inhibitor, an HDAC inhibitor, or a proteasome inhibitor.

12. 12. The method or composition of claim 11, wherein the immunosuppressant is an mTOR inhibitor.

13. 13. The method or composition of claim 12, wherein the mTOR inhibitor is rapamycin or a rapamycin analog.

14. The method or composition of any one of claims 1 to 13, wherein the viral vector is an AAV vector, such as an AAV8 vector.

15. 15. The method or composition of any one of claims 1 to 14, wherein the immunosuppressant drug loading is between 0.1% and 50%, on average across a population of synthetic nanocarriers.

16. 16. The method or composition of claim 15, wherein the immunosuppressant drug loading is between 1% and 30%, between 1% and 25%, between 1% and 20%, between 4% and 30%, between 4% and 25%, between 4% and 20%, between 8% and 30%, between 8% and 25%, or between 8% and 20%, on average across the entire population of synthetic nanocarriers.

17. The method or composition of any one of claims 1 to 16, wherein the synthetic nanocarriers are polymeric.

18. 20. The method or composition of claim 17, wherein the polymeric nanocarrier comprises a polymer that is a non-methoxy-terminated Pluronic polymer.

19. 19. The method or composition of claim 17 or 18, wherein the polymeric nanocarrier comprises a polyester, a polyester coupled to a polyether, a polyamino acid, a polycarbonate, a polyacetal, a polyketal, a polysaccharide, a polyethyloxazoline, or a polyethyleneimine.

20. 20. The method or composition of claim 19, wherein the polyester comprises poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), or polycaprolactone.

21. 21. The method or composition of claim 19 or 20, wherein the polymeric nanocarriers comprise polyesters and polyesters coupled to polyethers.

22. 22. The method or composition of any one of claims 19 to 21, wherein the polyether comprises polyethylene glycol or polypropylene glycol.

23. 23. The method or composition of any one of claims 1 to 22, wherein the mean of the particle size distribution obtained using dynamic light scattering of a population of synthetic nanocarriers is greater than 100 nm in diameter.

24. 24. The method or composition of claim 23, wherein the diameter is greater than 150 nm.

25. 25. The method or composition of claim 24, wherein the diameter is greater than 200 nm.

26. 26. The method or composition of claim 25, wherein the diameter is greater than 250 nm.

27. 27. The method or composition of claim 26, wherein the diameter is greater than 300 nm.

28. 28. The method or composition of any one of claims 23 to 27, wherein the diameter is less than 500 nm.

29. 28. The method or composition of any one of claims 23 to 27, wherein the diameter is less than 450 nm.

30. 28. The method or composition of any one of claims 23 to 27, wherein the diameter is less than 400 nm.

31. 28. The method or composition of any one of claims 23 to 27, wherein the diameter is less than 350 nm.

32. 27. The method or composition of any one of claims 23 to 26, wherein the diameter is less than 300 nm.

33. 26. The method or composition of any one of claims 23 to 25, wherein the diameter is less than 250 nm.

34. 25. The method or composition of claim 23 or 24, wherein the diameter is less than 200 nm.

35. 35. The method or composition of any one of claims 1-34, wherein the aspect ratio of the population of synthetic nanocarriers is greater than or equal to 1:1, 1:1.2, 1:1.5, 1:2, 1:3, 1:5, 1:7, or 1:

10.

36. For example, when the subject is a human, each lower dose of the viral vector may be 1e10 14 Less than 1e10 vector genomes / kg (e.g., 14 Each dose is at least 1 / 5, 1 / 5 or less, or 1 / 5 of vector genomes / kg or less than 5e 13 Less than 5e 13 36. The method or composition of any one of claims 1 to 35, wherein the amount of vector genomes / kg is at least 1 / 5, less than 1 / 5, or 1 / 5.

37. 37. The method or composition of any one of claims 1-36, wherein when the dosage or dosages of (i) comprise more than one dose of the viral vector, the doses of (i) of each dosage are administered over a period of 1 to 2 weeks.

38. 38. The method or composition of any one of claims 1-37, wherein when the dosage or dosages of (i) comprise more than one dose of the viral vector, the doses of (i) of each dosage are administered over a period of 1 to 3 months.

39. 39. The method or composition of any one of claims 1-38, wherein (ii) is administered in combination with a first dose of (i) for each dosage.

40. 40. The method or composition of any one of claims 1 to 39, wherein the subject is experiencing or has experienced a loss of transgene expression.

41. A lower dose of viral vector, 5e 13 41. The method or composition of any one of claims 1 to 40, wherein the vector genomes / kg or less.

42. A lower dose of viral vector, 2.5e 13 42. The method or composition of any one of claims 1 to 41, wherein the vector genomes / kg or less.

43. A lower dose of viral vector is 1–2.5e 13 43. The method or composition of any one of claims 1 to 42, wherein the total amount of vector genomes per kg is between 100 and 200 mg / kg.

44. A lower dose of viral vector, 1e 13 44. The method or composition of any one of claims 1 to 43, wherein the vector genomes / kg or less.

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